这项研究是根据俄罗斯政府的喀山联邦大学竞争力增长计划和俄罗斯基础研究基金会的资助（16-04-01051; 16-34-00817; 15-04-02983）进行的。我们感谢四位匿名审稿人对原稿草稿提供有用的意见。我们对Yuliya Aratskaya的录音表示感谢。我们非常感谢Victor Ilyin博士提供的许多有用的评论以及对手稿的最终编辑的帮助。

对来自蛙蛙蛙青蛙的肌肉皮肤 斑块的分离神经 - 肌肉制剂进行实验。两性的动物大小约5-9厘米。实验程序按照"喀山联邦大学和喀山医科大学实验动物使用指南"进行，符合NIH"实验动物护理和使用指南"。实验方案符合欧洲共同体理事会指令86/609 / EEC的要求，经喀山医科大学道德委员会批准。 1.准备解决方案<ol><li> 林格氏溶液的制备。 <ol><li>准备林格氏溶液:113.0mM NaCl，2.5mM KCl，3.0mM NaHCO 3和1.8mM CaCl 2 。将pH调节至7.2-7.4。 </li><li>用低Ca 2+制备林格氏溶液</高Mg 2+含量:113.0mM NaCl，2.5mM KCl，3.0mM NaHCO 3，6.0mM MgCl 2，0.9mM CaCl 2 。将pH调节至7.2-7.4。 </li></ol></li><li> 染料负载液的制备。 <ol><li>制备含有10mM（pH 7.2-7.4）的HEPES-Na的水溶液。 </li><li>将14μL的HEPES溶液加入到具有染料30的小瓶中。 注意:Ca 2+指示剂染料在500μL的小瓶中加入500μg粉末。 </li><li>涡旋旋转，彻底混合。 </li><li>稀释溶液使Ca 2+指示剂的最终浓度降至30mM。避免暴露于光线并储存在-20°C。 </li></ol></li></ol> 2.染色加载程序<ol><li> 用一块胸大肌神经解剖皮肤肌肉。 注意:解剖程序可用在Blioch 等人的文章的免费下载中，1968 31 。 <ol><li>对于解剖手术，使用两个精细钳子和角膜剪刀（参见表格 ）。将解剖的组织转移到预先填充有林格氏溶液的硅弹性体涂覆的培养皿中，并用精细的不锈钢针固定组织，使其在培养皿中略微拉伸。 </li><li>用新鲜的等份的林格氏溶液重新填充培养皿。取出结缔组织。不要伤害神经。 </li></ol></li><li>准备灌装移液器:使用剃刀刀片，切出标准塑料10μL移液管尖端的圆锥形部分〜2 mm长的一段。 </li><li>准备一块造型粘土，将填充移液管安装在培养皿上。 </li><li>将填充移液器的背面连接到塑料注射器，通过硅胶管和由移液器吸头制成的塑料连接适配器。 </li><li>之前d加载程序，使用塑料移液器从培养皿中取出林格氏溶液。使用精细注射器干燥肌肉 - 神经制剂;这将防止在随后加载灌装移液管时稀释Ca 2+染料。 </li><li>从冰箱中取出Ca 2+指示剂小瓶，并在室温下在黑暗的地方解冻。 </li><li>在立体显微镜控制下，低倍率（10×），检测肌肉和神经之间的连接。用精细的镊子和剪刀，将肌肉附近的胸骨神经切开（见步骤2.1）。留下2毫米长的神经残端。 </li><li>使用造型粘土将连接到管道和注射器上的注射器固定在培养皿上。 </li><li>移动吸头的尖端靠近神经残端。 </li><li>不夹住它，轻轻地将神经残端吸入灌装移液管的尖端。 </li><li>从白中取出吸管t填充移液器的末端。 </li><li>用长针头的注射器小心地从填充移液管中清除多余的溶液（参见材料表 ）。不要捏住神经残端。 </li><li>轻轻地垂直抬起填充移液管的尖端，使神经残端吸入尖端。 </li><li>使用凡士林，从灌装移液管尖端的外部绝缘吸入的神经残端部分。 </li><li>如果需要，可以将填充移液管中绝缘的神经残基干燥:使用长针头注射器从灌装移液管中轻轻吸出过量的溶液。 </li><li>使用长吸液管吸头吸取0.5μL染料加载溶液（参见步骤1）。 </li><li>轻轻地将移液管尖端与装载溶液插入到填充移液器中。将混合物直接喷到神经残端上。 </li><li>用凡士林密封灌装移液器的开口端。 </li><li>添加一小块林格的注意培养皿以保持准备湿润。 </li><li>在黑暗和潮湿条件下室温孵育5小时。 </li><li>用加载溶液取出填充移液器，用林格氏溶液冲洗准备物质，并在8°C的冰箱中保持过夜。 </li></ol> 3.准备显微镜组织<ol><li>将制备物装入硅弹性体涂层室中，并用钢微针固定，使其略微拉伸。 </li><li>用新鲜的林格氏溶液等份冲洗组织。 </li><li>使用吸电极刺激神经;电极的构造可从Kazakov 等人免费下载。 ，2015 32 。将电极尖靠近神经的切割端并将神经残端吸入电极孔。 </li><li>将准备室安装在显微镜平台上。的Plac温度探头和腔室中的入口和出口点火。 </li><li>将电源线连接到珀耳帖元件。 </li><li>为了超级准备，请使用简单的重力驱动系统。要清除多余的溶液，请打开灌注抽吸泵。 </li><li>打开热控制器单元。 </li><li>将温度控制设置为20°C。 </li><li>安装紫外线防护罩。 </li><li>将刺激线电极连接到电刺激器，并用4x物镜观察显微镜下的肌肉收缩。 </li><li>用低Ca 2+和高Mg 2+含量的林格氏溶液填充灌注系统。 注意:该解决方案用于预防肌肉收缩。外部钙浓度的降低和外部镁的升高导致Ca 2+瞬变幅度的降低。然而，根据以前的经验，将0.9mM CaCl <sub&gt; 2和6mM MgCl 2仍足以可靠地分辨Ca 2+瞬变的幅度。值得一提的是存在一些减少肌肉收缩而不降低Ca 2+浓度的方法。例如，使用d-tubocurarine或α-银环蛇毒素，特异性阻断烟碱乙酰胆碱受体将完全或部分阻断肌肉抽搐17，27，28，30。然而，这些毒素的添加也可以影响突触前钙进入33 。为了避免这种情况，可以使用μ-芋螺毒素GIIIA。 </li><li>打开泵，并用低Ca 2+和高Mg 2+的林格氏溶液启动制剂的灌注。 </li><li>在显微镜上切换到40×物镜。 </li><li>打开t<html>他单色仪（见材料表 ）。 </li><li>在单色仪控制软件中选择488 nm的发射波长和连续的照明模式。 </li><li>在高放大倍数的荧光模式下，确保神经末梢装载染料。 </li></ol><img alt="图1" class="xfigimg" src="/files/ftp_upload/55122/55122fig1.jpg" /> 图1 :带有Ca 2+指示剂的神经和终端。 （ A ）装载程序后，神经中装有Ca 2+指示剂。刻度棒= 200μm。 （ B ）充满Ca 2+指示剂的神经末梢。刻度棒=20μm。 （ C ）Ca 2+指示剂荧光在神经末梢清晰可见。刻度棒=20μm。_blank"&gt;请点击此处查看此图的较大版本。 <ol start="17"><li>允许制剂在低Ca 2+和高Mg 2+溶液中平衡至少30分钟。 </li></ol> 4.采用数字CCD相机进行视频采集注意:捕获荧光信号的细节对于每种显微镜和相机类型都是特定的，但关键考虑的是图像捕获速度。 <ol><li>使用1 kHz作为记录NMJ中单个Ca 2+瞬变的最小捕获频率。 注意:快速数字CCD摄像机是荧光成像所必需的（见材料表 ）。数据采集​​系统和软件（参见" 材料表" ）用于摄像机，单色仪和刺激器的同步。简而言之，该协议允许在数据的数字输出上产生同步脉冲采集系统打开快门，捕获视频信号，并启动刺激。可以在协议和/或设备上设置所有时间参数。一个典型的协议是一系列以1 kHz（80 x 80像素）获得的500帧。用激发光照射可以漂白Ca 2+指示剂并对细胞组织造成光损伤。因此，避免激发光的长时间曝光。在此协议中，快门仅在拍摄视频所需的时间打开。每个特定神经末梢获取二十个系列。目的是监控对照组和给药后的相同部位。 </li><li>在显微镜的4X物镜下，使用亮场方式来显现肌肉和神经分支。 </li><li>切换到40X物镜，使用落射荧光方式和488nm的激发波长，寻找负载染料的神经末梢。确定感兴趣的神经末梢区域。 </li><li>在三目管of显微镜，选择光路交换等级:100％照相机。 </li><li>启动CCD摄像机的采集软件。 </li><li>在"实时"模式下，找到ROI并调整焦点。 </li><li>选择菜单"更改"设置。 </li><li>使用"基本配置"，每秒1000帧（fps），分辨率为80 x 80。 </li><li>将输入帧数设置为500。 </li><li>输入实验的名称。 </li><li>选择"外部触发"。 </li><li>将预触发时间设置为10 ms。 </li><li>将重复次数设置为20。 </li><li>在单色仪控制软件中，选择488 nm的发射波长和"外部触发照明"模式。 </li><li>运行数据采集软件。 </li><li>加载刺激方案。 </li><li>在录制视频之前，请使用视频采集软件捕获暗帧。 </li><li>运行刺激方案。 </li><li>选择ROI和check记录的信号。 </li></ol>数据分析 NoOTE:对于数据分析，请使用CCD相机软件和ImageJ;数据表示为电子表格程序中的曲线。在CCD相机软件中，平均20次重复并将结果导出到ImageJ支持文件。在ImageJ中，选择ROI和背景。从ROI中减去背景。将数据表示为:（ΔF/ F 0 -1）×100％，其中ΔF是刺激期间的荧光强度，F 0是静止时的荧光强度。 <ol><li>在CCD摄像机的采集软件中，单击文件&gt;平均文件。选择文件并对其进行平均。 </li><li>将平均文件保存为.fit文件，方法是单击"另存为文件"。 </li><li>运行ImageJ软件。执行以下步骤: </li><li>点击图像&gt;调整&gt;亮度/对比度。 </li><li>单击图像&gt;堆栈&gt;工具&gt;;堆垛机</li><li>点击分析&gt;工具&gt;投资回报率管理器。 </li><li>将平均的.fit文件拖放到ImageJ窗口中。 </li><li>放大窗口以获得更好的视野。 </li><li>通过移动光标，选择最后一帧并将其删除（这是黑框） </li><li>在被认为是背景的区域上选择一个矩形ROI。将其添加到ROI管理器</li><li>单击更多&gt;多尺度测量背景。注意平均值。复制数据，将其导出到电子表格程序，并计算一个比率的阈值的平均值。 </li><li>通过单击"处理"&gt;"主"&gt;"减"，减去堆栈中的阈值。输入阈值的平均值。 </li><li>在神经末梢周围选择一个矩形ROI。将其添加到ROI管理器。 </li><li>单击更多&gt;多尺度测量。注意平均值。复制并将其导出到电子表格程序。 </li><li>平均信号的偏移。 注意:使用第一几十点显示基色染料无刺激;这是休息时的荧光。 </li><li>将信号除以休息时的荧光。 </li><li>减去"1"乘以100％。 </li><li>绘制信号并计算Ca 2+瞬变的幅度。 </li></ol>

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K., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraterminal+Ca2++and+spontaneous+transmitter+release+at+the+frog+neuromuscular+junction.">Intraterminal Ca2+ and spontaneous transmitter release at the frog neuromuscular junction.</a> J Neurophysiol. 85 (1), 287-294 (2001).</li><li>Shahrezaei, V., Cao, A., Delaney, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++from+one+or+two+channels+controls+fusion+of+a+single+vesicle+at+the+frog+neuromuscular+junction.">Ca2+ from one or two channels controls fusion of a single vesicle at the frog neuromuscular junction.</a> J Neurosci. 26 (51), 13240-132499 (2006).</li><li>Troncone, L. 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作者没有什么可以披露的。

在本文中，我们介绍了通过神经残端对青蛙神经末梢进行Ca 2+敏感染料负载的方法。在装载程序结束时，神经近端部分的所有端子都具有显着的荧光水平。已经估计探针的端内浓度在40和150μM之间变化17 。 孵育过程分两步进行:在室温下，然后在较低温度下在冰箱中进行。在室温下控制染料组织培养的时间很重要。根据神经残端的实际长度，特定染料和温度，孵育时间可能会有所不同。如果曝光过度，靠近神经残端的近端部位的终端可能过载。然而，在神经的中间部分，仍然可以找到令人满意的载物。期间在冰箱中长时间保温，染料均匀分布于神经末梢。 我们自己的观察33,35以及其他研究人员的数据30证明了加载过程对突触后反应的振幅或微型端板电位的频率没有任何明显的影响。在装载的准备中记录了良好的寿命。我们想提请注意一些重点。在切除后几分钟内将神经残基置于染色剂加载溶液中是非常重要的，以使染料进入切割神经的轴突;延迟可能导致无效的负载，大概是由于神经轴突27,36的重新密封。一些研究者将神经残体浸入100mM EDTA（Ca 2+和Mg 2+螯合剂）中）切除神经后立即防止切口轴突重新密封。 1-2分钟后除去缓冲液，用染料加载溶液37代替。使用石油井代替塑料管用于装载程序允许使用较短的神经残端。在使用这种方法时，在用染料浸入HEPES溶液中之后切断神经，并且由于染料溶液27,28中缺少二价离子，轴突不会重新密封。 在我们的研究中，我们使用Ca 2+指示剂的水溶性盐形式而不是葡聚糖。葡聚糖结合物在轴突中比盐形式更慢地扩散。然而，使用葡聚糖缀合物减少了神经和NMJs的染色分隔和处理。钙绿色1-3,000MW葡聚糖共轭物具有良好的扩散速率并表现出减少的区室化<s<html> up class ="xref"&gt; 38。 避免组织长时间的荧光照射是非常重要的，因为这会影响组织的健康和生存。我们在可见光通道中使用Nomarski光学器件来搜索神经末梢。在录制过程中，我们使用隔膜来限制照明场。 值得注意的是，这种加载方法仅适用于可以承受长时间孵化的制剂。为了减少对较脆弱的组织（ 例如温血动物的突触）进行研究时的染色加载时间，有必要缩小神经残端长度并使用微量移液管装载29,39。 这种加载技术非常适合于细胞溶质Ca 2+的成像变化，具有单神经刺激和节律突触活动下的荧光指示Ca 2+ -瞬态振幅的分析可用于研究不同物质对参与神经递质释放的钙进入的影响33 。

钙离子（Ca 2+ ）参与许多神经元信号过程，包括介体释放1,2,3,4,5的起始，维持和可塑性。当动作电位到来时，细胞外Ca 2+进入神经末梢并启动神经递质释放。在一些突触中，钙电流可以通过电生理方法6,7,8直接测量。在神经肌肉接头（NMJ）的情况下，由于神经末梢的微小尺寸，不能使用直接膜片钳和双电极电压钳技术。 NMJ神经末梢的内向Ca 2+电流的记录可以通过间接电泳进行生理方法9,10 。然而，这些方法需要由钠和钾离子通道阻断剂预处理突触。光学方法不需要神经末梢中的离子电流的药理学分离，并允许由动作电位触发的Ca 2+内流的记录，以及随后在轴突质体11,12,13,14中升高Ca 2+离子。这些方法基于在游离Ca 2+离子15,16,17,18,19的结合时记录特定Ca 2+敏感染料的荧光变化。 Ca 2+指示剂可以加载到c通过各种方法，取决于实验的目的。研究人员使用通过贴片移液管22或显微注射23,24,25加载的透膜性染料形式20,21的浴液。然而，所有这些方法在NMJ的情况下都有一些限制，因为它在突触建筑学中的特性。对于NMJ，最方便和最成功的方法是将染料载入神经残端，一种正向填充法26,27,28,29 。该技术可用于将各种荧光染料加载到周围神经末梢。该方法成功应用于果蝇神经末梢28 ，蜥蜴运动神经28 ，青蛙运动神经终端17,26,27,30 。根据研究对象，有条不紊的细节可能会有所不同。玻璃微量吸管可用于来自幼虫28的小神经。一些研究人员已经描述了一种方法27,28 ，其中将肌肉神经支配的新鲜切割的末端浸入预充满染料的孔中。然后将制剂放置几个小时以浸泡染料。染料被轴突浸泡并运送到神经末梢。在本文中，我们描述了通过神经残端将荧光指示剂加载到青蛙运动神经末梢的方法。我们的协议使用塑料移液器吸头来孵育组织和染料。我们还描述了如何获取和分析Ca 2+荧光transients。

<table><tbody><tr><td>Ca2+ indicator</td><td>Molecular Probes, USA</td><td>Oregon Green 488 BAPTA-1 hexapotassium salt, O6806</td><td>500 &amp;mu;g</td></tr><tr><td>Silicone Elastomer</td><td>Dow Corning, USA</td><td>Sylgard 184 elastomer</td><td></td></tr><tr><td>Pipette</td><td>Biohit, Russia</td><td>720210</td><td>0.5-10&amp;micro;L</td></tr><tr><td>Pipette tip</td><td>Fisher Scientific, USA</td><td>02-707-175</td><td>10&amp;micro;L</td></tr><tr><td>Pipette tip</td><td>Biohit, Russia</td><td>781349</td><td>10&amp;micro;L</td></tr><tr><td>Razor Blade</td><td>Fisher Scientific, USA</td><td>12-640</td><td></td></tr><tr><td>Minutien Pins</td><td>Fine scince tools, Canada</td><td>26002-20</td><td></td></tr><tr><td>Corneal Mini-Scissors</td><td>MT MEDI CORP, Canada</td><td>S-1111</td><td></td></tr><tr><td>Jeweler Forceps</td><td>MT MEDI CORP, Canada</td><td>F-9610</td><td></td></tr><tr><td>Jeweler Forceps</td><td>MT MEDI CORP, Canada</td><td>F-9611</td><td></td></tr><tr><td>Modelling clay</td><td>local producer</td><td></td><td>can be replaced by any local producer</td></tr><tr><td>Petroleum jelly</td><td>local producer</td><td></td><td>can be replaced by any local producer</td></tr><tr><td>Microspin FV 2400&amp;nbsp;</td><td>Biosan, Latva</td><td>BS-010201-AAA</td><td></td></tr><tr><td>Multi-spin MSC 3000</td><td>Biosan, Latva</td><td>BS-010205-AAN</td><td></td></tr><tr><td>Single-use hypodermic needles</td><td>Bbraun</td><td>100 Sterican</td><td>0.4&amp;times;40mm</td></tr><tr><td>Syrynge</td><td>local producer</td><td></td><td>0.5 ml</td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma-Aldrich, USA</td><td>H0887</td><td>100ml</td></tr><tr><td>Microscope, BX51</td><td>Olympus, Japan</td><td></td><td></td></tr><tr><td>Data acquisition system&amp;nbsp;</td><td>Molecular Devices, USA</td><td>Digitdata 1550</td><td></td></tr><tr><td>software</td><td>Molecular Devices, USA</td><td>pClamp software, Version 10</td><td>protocol can be download from : http://kpfu.ru/portal/docs/F_230007060/Video.capture.with.&lt;br/&gt; RedShirt.Neuro.CCD.camera.pro</td></tr><tr><td>Bath and bath temperature controler</td><td>Experimental Builder</td><td></td><td>can be replaced by any chamber with temperature control. For example from https://www.warneronline.com/&amp;nbsp;</td></tr><tr><td>Monochromator</td><td>Till Photonics, Germany</td><td>Polychrome V</td><td>no longer available, can be replaced by other&amp;nbsp;&amp;nbsp; sutable stable light source 488 nm</td></tr><tr><td>monochromator control software</td><td>Till Photonics, Germany</td><td>Polycon</td><td></td></tr><tr><td>Digital CCD camera</td><td>Redshirt imaging, USA</td><td>Neuro CCD SMQ</td><td></td></tr><tr><td>Model 2100 Isolated Pulse Stimulator</td><td>A-M Systems, USA</td><td></td><td></td></tr><tr><td>Acquisition software for CCD camera</td><td>Redshirt imaging, USA</td><td>Turbo SM software</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health, USA</td><td></td><td>http://rsb.info.nih.gov/ij/download.html</td></tr><tr><td>Spreadsheet program</td><td>Microsoft, USA</td><td>Microsoft Office Excel&amp;nbsp;</td><td></td></tr></tbody></table>

loading a calcium dye into frog nerve endings through the nerve stump: calcium transient registration in the frog neuromuscular junction

测量突触前神经末梢突触前钙水平的最可行方法之一是光学记录。它基于使用钙敏感的荧光染料，其根据细胞中游离钙的浓度改变其发射强度或波长。有几种方法用钙染料染色细胞。最常见的是通过微量移液管装载染料或与染料的乙酰氧基甲酯形式预孵育的过程。然而，由于出现的方法问题，这些方法不太适用于神经肌肉接头（NMJ）。在本文中，我们提出了一种通过蛙神经的青蛙神经残基将钙敏感染料载入神经末梢的方法。由于外部钙进入神经末梢并随后与钙染料的结合发生在毫秒级时间内，所以有必要使用快速成像系统来记录这些相互作用纳秒。在这里，我们描述了一种使用快速CCD相机记录钙瞬态的协议。

染色剂负载后，运动神经刺激后，可以在神经末梢检测到荧光信号（Ca 2+瞬态）振幅的增加（ 见图2 ）。 Ca 2+瞬变的参数如表1所示 。从数量上看，本研究中测量的Ca 2+瞬态参数与其他科学家在冷血动物15,34的突触中获得的数据接近。 Ca 2+瞬变的参数取决于Ca 2+与染料的结合速率和随后的解离。 Ca 2+进入神经末梢，与染料的相互作用和细胞质扩散的速率都影响Ca 2+瞬时的上升时间。荧光信号的衰变时间取决于染料的亲和力，Ca 2+与细胞内缓冲液相互作用的速度以及离子泵35的去除。 Ca 2+瞬变的振幅分析可用于研究各种物质对参与神经递质释放的钙进入的影响33 。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55122/55122fig2.jpg" /> 图2 :平均Ca 2+ 在青蛙NMJ中进行瞬态测量。基于来自13只青蛙NMJ的信号的平均值计算Ca 2+瞬变。 <a href="//ecsource.jove.com/files/ftp_upload/55122/55122fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 峰ΔF/ F（％） </td><td> 上升时间20％-80％（ms） </td><td> τ（ms） </td></tr><tr><td> 12,6±1,1（n = 13） </td><td> 4.6±0.5（n = 13） </td><td> 115.3±8.3（n = 13） </td></tr></tbody></table> 表1: Ca 2+瞬态 的 平均 参数 。数据表示为平均值±SE; n是不同NMJ中的测量次数。峰值ΔF/ F是ΔF/ F的平均振幅。

Watch this Scientific Journal Video about Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction at JoVE.com

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

在这里，我们描述一种通过青蛙神经残端将钙敏感染料载入神经末梢的方法。我们还提出了记录和分析周围神经末梢快速钙瞬变的方案。

通过神经树桩将钙染料加载到青蛙神经末梢:青蛙神经肌肉接头中的钙瞬态登记

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

loading-calcium-dye-into-frog-nerve-endings-through-nerve-stump

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

7.7K 次观看.  Russian Academy of Sciences. 在这里，我们描述一种通过青蛙神经残端将钙敏感染料载入神经末梢的方法。我们还提出了记录和分析周围神经末梢快速钙瞬变的方案。 

视频: Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

载入果蝇与神经末梢钙指标

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

科研

生物学

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

生物化学

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

监测变化的细胞内钙离子浓度和突触效能的软体动物<em&gt;海兔</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

神经科学

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 ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

荧光钙成像及其后续原位杂交，用于异种性前体特征

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

发育生物学

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

调频染料循环在突触: 高钾偏极化, 电和 Channelrhodopsin 刺激的比较

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

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 ...

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

使用共聚焦显微镜以高时间分辨率记录小鼠神经肌肉接头中的钙瞬变

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...

通过神经树桩将钙染料加载到青蛙神经末梢:青蛙神经肌肉接头中的钙瞬态登记

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