这项工作是由来自加拿大自然科学和工程研究理事会（NSERC）和加拿大创新基金会（CFI）为DWA资助。

1。准备和解剖<ol><li>准备内衬的Sylgard的解剖盘。从WPI（萨拉索塔，佛罗里达州，美国;供应商见表2）录音室被用作解剖菜（ 图1）。该室的设计，以适应玻片和小塑料垫圈被用作模具的的Sylgard。该垫圈是第一固定用油脂和液体的Sylgard被加入到洗衣机的中心滑动。该的Sylgard可以治愈过夜，因为它确实是这样，它形成了一个小的，圆床于载玻片上，它成为解剖盘的基础。滑动被放置到该记录室中，并作为所述腔室至该制剂被固定（ 图1）的基极。 </li><li>制备表3中列出的解决方案。细胞外溶液应在室温下进行，同时留下胞内溶液应保持无菌。你也可以添加卢cifer黄色（0.1％）至细胞内的溶液中，以便中的M-细胞形态学该可视化可以在实验结束时进行。这是为了确认小区标识（ 图2）。 </li><li>在提高水蛋野生型斑马鱼胚胎在28.5℃，根据金梅尔等 10收集和舞台。对于解剖，胚胎转移到解剖盘，它还可以作为录音室。其值接近生理盐水;在麻醉盐溶液（溶液1， 表3 0.02％三卡因）麻醉7-10分钟。人们可以判断自己是否通过检查响应温柔的尾巴捏充分麻醉。 </li><li>通过脊索和到的Sylgard林立的菜0.001"钨丝用25X的放大倍率在解剖显微镜（ 图1）（科学仪器服务。公司，RINGOES，NJ，USA）。钨丝很容易切脚胚胎在与精细解剖剪刀小片段。 </li><li>调整的解剖范围的放大倍率为40-50X进行解剖。一旦胚胎被固定的菜，下巴，眼睛和脑用细镊子（杜蒙＃5）被删除。 </li><li>后脑被仔细地工作脊索和大脑之间的镊子轻轻剥离从底层的脊索。后脑，然后轻轻地翻转过来，使腹面朝上。这种定位提供了良好的前往M-细胞，这是通常在5-10微米腹面的幼胚和〜20-50微米的中老年幼虫（ 图2A）。 </li><li>编制小心地移动到可以进行膜片钳实验的录像设置。对M-细胞可视化与利用Nomarski微分干涉对比（DIC），虽然成像的其他方式从（ 即霍夫曼调制对比或DODT梯度造影Luigs和诺依曼，德国），也可以使用。 </li></ol> 2。膜片钳记录（全细胞模式） <ol><li>所有记录均在全细胞膜片钳模式11进行。录音室被放置到一个显微镜载物台在电设置。我们的阶段是固定的，直立的膜片钳显微镜用螺栓固定到一个可移动的显微镜平台或显微镜的翻译程序。 </li><li>膜片钳移液器被拉上水平拉出器（P-97;萨特仪器有限公司）从WPI获得薄壁，硼硅酸盐玻璃。移液管尖端直径为0.2-0.4微米的量级火抛光至光滑的边缘后，与柄锥度为〜4毫米的长度。 </li><li>附上吸管给放大器头段中的电设置。它保持头部侧以大致45°角的水平轴，因为这确保了移液管是适合的高电阻密封形成一个入口角是很重要的S里的毛特纳细胞。 </li><li>刚进入槽液之前，将少量的正压，以吸管，以减少弄脏尖端的机会。当录制mEPSCs，沐浴解决方案包括1微米TTX阻断动作电位，5微米士的宁阻断甘氨酸受体和10μM荷包牡丹碱或100微米印防己毒素阻断GABA A受体。当录制的mIPSC，沐浴解决方案包括1微米河豚毒素，犬尿喹啉酸，要么荷包牡丹碱或马钱子碱根据感兴趣的受体活性。 </li><li>接近的M-细胞用少量的在吸液管的正压力。正压轻轻推压从侧面小区到另一侧，当在细胞立即定位，形成于细胞膜上的小凹坑。留在原地移液器几秒钟轻轻擦拭细胞表面，从而能够形成在吸液管和隔膜之间的强密封。释放在移液管的正压力允许密封地发起和少量的负压力加上负移液管电位GigaΩ密封在几秒钟内形成的效果。 </li><li>更改放大器-60 mV的控股潜力。破裂细胞膜具有一系列抽吸的短脉冲。立即记录膜电位，并尽量减少电容的文物。由70-85％补偿细胞电容（C M）和访问（系列）电阻（R 一 ）。 R A，应定期监测，每30秒到一分钟，并且如果有20％或更多的改变，中止实验。 </li><li>一旦实验结束和足够的数据已被获取，该制剂是通过用一对镊子去除后脑处死。在这一点上，数据分析就可以开始。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

以上所述的协议允许一个从M-细胞和其同系物的膜片钳记录。该技术是具有挑战性的，并应注意在整个过程中采取的几个关键领域。现在，我们将讨论一些这些方面的，并会提供有用的提示，以确保成功。 解剖/录音室的结构是本实验的一个重要方面。对M-细胞是很难看到，除非微分干涉对比使用。利用Nomarski DIC效果最好用干净的玻璃盖玻片上，但是我们已经发现，非常薄的层的Sylgard（〜1-2毫米），不会扭曲DIC在于，它与识别的M细胞的干扰的程度。因此，我们使用一个记录腔室具有在底部有一个薄的玻璃盖玻片上，其上放置了一个小的塑料垫圈（〜5毫米直径）。我们在培养皿中混合的Sylgard 184和仔细的液体的Sylgard加至用巴氏吸管将垫圈的内侧。在施lgard可以在室温下固化超过几天或可加热的快速固化。垫圈可后的Sylgard固化去除，尽管这不是必需的。一旦剥离/记录室已经作出，它可以用于几个星期一个新鲜的Sylgard内衬载玻片之前需要。 此过程中的下一步是弥补所有的实验（ 表3）当日的相关解决方案。包含的一切，但ATP和GTP的细胞内溶液在-20℃下制成提前并存储在5ml等分上记录的一天的等分试样解冻至室温，新鲜的ATP和GTP被添加。将溶液调节至280毫渗透摩尔浓度和pH为7.4。在我们手中，我们发现，除了新鲜的ATP和GTP每天产生良好的录音。所有的解决方案需要保持无菌成功的最佳机会。 细胞内的解决方案必须经过过滤throug公顷0.22微米的过滤器（Sigma公司），以去除小颗粒和碎片，可能会影响录音。要做到这一点，我们首先绘制细胞内溶液倒入1毫升注射器，加盖过滤器，注射器的末尾，添加一个塑料吸管尖端的加热并拉至精尖，到过滤器的末尾。这使得我们可以直接将过滤后的细胞内溶液的玻璃补丁移液器的内部。 一旦解决方案准备，胚胎可被麻醉，解剖。在此之前的夹层，应注意充分评估生物体的时代。在离合器年龄每个胚胎在一个稍微不同的速率。因此，评估生物的年龄时，这样做的根据既定程序10,13，通过利用表型线索，如体节的数量，身体的整体形状和卵子受精的时间是很重要的。所有的解剖与一个杜蒙＃5细对执行镊子。这些需要是尖锐，处于良好的工作状态，否则这将是非常难以去除的皮肤覆盖在后脑的腹面的后脑和任何结缔组织。我们经常要锐化镊子几个星期的使用后，做自己该用磨石。 到脚鱼薄层SYLGARD的，我们用针从细钨丝0.001英寸直径塑造。该引脚切断与旧，显微解剖解剖显微镜下剪刀，并有足够的小，以适应的权利，通过脊索。钢钉胚胎在任何其他点不固定它们，使夹层几乎不可能完成。当第一次学习来执行记录，也可以是难以从它的同系物的同一性的M-细胞（特别是MID2厘米，位于相邻的菱 - 菱5）。在某些胚胎这两对细胞的出现在大小相似。耳石亲韦迪一个方便的地标在M-细胞的识别提供帮助。在48 HPF的M细胞位于旁边的耳石，即使耳石解剖过程中删除，他们的后脑轻微受损的横向区域，作为一个标记为原来的位置，身后离开。表达GFP或一些其它荧光标记物中的M-细胞的转基因鱼，提供优良的准备新实验者。在我们手中的M-细胞具有约18-24 pF的膜电容在48个高倍视野，而较小的同系物是接近12-14 pF的。较小的细胞相比，细胞具有更大的表面积有较大的膜的电容值（在微微法拉（PF）测量）。因此，膜电容可以作为泡孔尺寸，其中较大的细胞具有更大的电容值的粗略指标。此电特性作为另一属性由该M-细胞可以从它的同系物进行识别。最后，我们经常使用鲁cifer黄色在我们的记录（细胞内）解决方案，它允许我们使用荧光成像一次录音是完全受调查进行坚决鉴定细胞类型。 在3.5-4.5MΩ范围的枪头电阻是烙铁头尺寸和低接入电阻，一般为8〜15MΩ之间的最佳折衷。这是为了与快速动力学事件尤其重要;〜0.1毫秒（20-80％）的上升时间和〜0.5毫秒呈指数衰减12,14,15（ 图3A和3B）。获取在50-100 kHz的数字化速率数据，并过滤以5-10千赫的低通频率。关于补丁移液器，我们发现，他们并不需要是火抛光，以得到记录，但有趣的是，它似乎提供稍微好一点的机会，取得了良好的密封与糙米移液器相比。由于移液器吸头比较大，我们不加版本Ÿ多正压吸管进入解决方案之前。这需要练习后决定的正压申请成为过剩的压力适量将移动单元格太多，而且可以防止密封形成。它也可能损害突触接触的细胞轻轻地从它的原始位置移动。在另一方面，压力太小会造成脏技巧和不干净的细胞不够好，形成良好的密封表面。一个折中的仅是显著的实践和经验获得。密封的形成可以与负电压，以移液管的应用得到加强。我们通常有1.2-2GΩ的印章，这是极好的突破到全细胞模式。 当药物制剂需要被施加到细胞的细胞质中，我们将它们包括在填充所述吸移管前细胞内的溶液。护理应准备吸管的解决方案时，避免药物的损失采取由具有其绑定到0.22微米的过滤器。因此，我们首先筛选的细胞内介质，然后在药理学试剂仔细添加到过滤后的溶液。化合物在细胞内的隔间应用程序都有自己的一套独特的挑战。举例来说，除了药物对细胞内的介质通常降低形成GΩ封的机会，而不是到这种地步，这是一个重大障碍的实验。 人们应该认识到在这种方式药理剂的应用程序不允许实验者记录的最合适的控制，因为该剂具有从全细胞组态的起始立即进入细胞内。因此，尽管我们在本类型的实验记录数据，人们必须意识到，下一个最好的控制是代理的非活性形式在细胞内的应用。在许多情况下，这需要一个热灭活化合物的形式时，加扰的肽ør是供应商获得一个无效的异构体。 我们获得了在突触电流（MPSCS），电压门控电流和动作电位的表单数据。微型电流可能由几个优秀节目（pClamp，Axograph等）进行分析，虽然我们优选使用Axograph X（约翰克莱门特）。 Axograph X运行于Windows和Macintosh平台上，可用于电生理数据的同时采集和分析。 mEPCs购入用实验者的选择一个模板，从所述记录本身得到的援助。单个事件可以为属性，如上升时间，幅度，半宽度和衰减时间，等我们的数据的电子表格导出为Kaleidagraph科学（协同软件），我们可以生产数字，包括Axograph X的事件跟踪副本进行分析一个实验的更困难的方面是确定是否执行不够好，以提供清洁的记录，可靠的数据。例如，空间夹紧问题可能记录从M-细胞，具有较大的轴突和树突的广泛过程MPSCS时出现。当电压钳位，可以大致控制电压在移液 ​​管的尖端相当好（特别是如果所述接入电阻（R a）为低），但可能没有足够的膜电位的适当的控制中树枝状突起或轴突，远远远离枪头。确定所述细胞为正常空间夹紧的一种方法是产生的上升时间的散点图与被记录的MPSCS的振幅。数据之间的相关性是暗示空间夹紧不足。换句话说，如果该空间钳较差然后发生靠近吸管MPSCS将具有更快的上升时间和更大的幅度比存在一段距离的移液管的事件。如果一个相关性不存在，则该数据是有问题的，并且不能被使用。 另一个方面电生理记录，需要加以解决的是，泄漏电流。这一点尤其是当电压钳位，以便记录电压-门控电流的如Na +或K +电流的小区。当膜电位从其余偏离时，泄漏电流可以随着电压 - 门控的​​活动被记录。泄漏电流（I L），钾（ⅠK）的组合，钠（I Na）的氯化物和（I Cl）的电流通过非电压门控通道将掩盖感兴趣的活性，必须从记录中减去。该放大器能够运行什么通常称为P / N协议的录制过程中在线执行此功能。在一个P / N协议放大器将来自其余运行几个小的去极化（或hyperpolarizations），并会记录发生所产生的漏电流。使用足够小的脉冲不激活电压门控通道是很重要的。当前在这些脉冲中发生s的记录，并取平均值，以便这些泄漏电流可以从电压 - 门控通道活性被减去。 最后，一​​个需要考虑的结电位，其中发生在胞内和胞外溶液之间的电极的尖端。细胞内和细胞外溶液通常由不同的离子，具有不同的活动和迁移率。较大的离子具有较低的迁移率将提供一个较大的电阻率，以离子运动比小，这可能导致在溶液的结的小，但显著电位差。确定所经历的细胞的适当的电压时，此结电位，必须考虑到。 这里介绍的技术是一家长年使用我们的实验室。但是，也有记录的从M-细胞的替代方法。最值得注意的是，约瑟夫Fetcho和他的同事开发编极好的制剂，其中一个可以从旧的幼虫（4-5日龄）的一种微创方式的M-细胞比在此处，M-细胞是从背侧表面16接触提交记录。我们曾尝试了类似的技术，但发现它更容易从后脑，其中所述细胞是更接近大脑的表面，尤其是在年轻鱼（ 即 24个高倍视野至72 HPF）的腹侧面接近的M-细胞。另外，从背表面上的方法通常需要使用表达荧光标记的M-细胞，其具有引入额外的变量到记录的电势的转基因系的。当使用野生型鱼这个问题不存在。然而，这里介绍的技术，我们只限于研究年轻的动物（24个高倍视野72个高倍视野）。因此，每一种方法都有其优点和局限性。 学生氏t-检验，可用于比较两个组数据而瓦里安的分析CE（ANOVA），可用于比较多个组。必须小心选择合适的事后检验的参数与非参数数据。

其中一个在发育神经生物学有关的未决问题涉及到的突触可塑性现象在突触发育的作用。我们的研究重点是了解背后的突触发展的理解突触发展的背景下动物行为的总体目标可塑性的机制。为了做到这一点，我们开发了一个基本完好制剂生物（斑马鱼， 斑马鱼 ），已经获得了相当大的牵引发育研究在90年代末。 斑马鱼提供了神经发育研究中几个明显的优势。比如，它的基因组测序和一个可以执行吗啉击倒和锌指核酸酶敲除沉默基因活性和蛋白表达。一个也可以执行过表达研究和转基因品系可以构造7-9。胚胎是相对容易和丰富的收购，而胚胎的透明性质非常适用于成像和光遗传学研究。此外，行为分析可以被执行，和电生理实验，可以进行对肌肉纤维，脊髓神经细胞和神经元后脑在基本完好的生物体，允许一个检查体内的细胞功能。重要的是，我们能够研究突触活动中不仅同一类细胞，但在非常相同的一对神经元，从制备制剂。换句话说，这是罕见的脊椎动物制剂之一，其中一个可以返回到相同的神经元， 在原地 ，以研究其发展。 为了维持神经回路作为可行的可能，我们开发了一种制备其中的后脑和脊髓被原封不动，而膜片钳从M-细胞。在此制备过程中，眼睛，下巴和皮肤覆盖在大脑轻轻取出，后脑是皮尔ð远离脊索，允许访问后脑腹侧表面。该reticulospinal神经元位于几微米深，都比较方便。在48小时后的受精（HPF;一些缩写列于表1）的细胞进行电紧凑，可以忠实地记录突触活动（包括兴奋性和抑制电流），电压-门控电流和动作电位从它们。 我们的研究结果表明，突触发育和成熟的许多方面孵化前发生的24小时，因此备受大家关注的重点是生物年龄从24个高倍视野72个高倍视野。然而，由72个高倍视野，M-细胞是少电紧凑，而且难以正确地空间钳。该技术可用于从M-细胞，而且从它的同系物，MID2厘米和MID3厘米不仅记录。理论上，也可以执行双重录制，从SPINAL脊髓神经细胞如初级运动神经元和来自M-细胞，但这是困难的，它可能被认为由这样的困难的技术获得的利益可能不值得所需要的非凡的努力。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Equipment Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		 </tr>
		 <tr>
			<td>Dissecting Dish</td>
			<td>Warner Instruments</td>
			<td>64-0291</td>
		 </tr>
		 <tr>
			<td>0.001" Tungsten Wire</td>
			<td>Scientific Instruments Services Inc.</td>
			<td>W406</td>
		 </tr>
		 <tr>
			<td>Dissecting microscope</td>
			<td>Leica Microsystems</td>
			<td>MZ9.5</td>
		 </tr>
		 <tr>
			<td>Pipette Puller</td>
			<td>Sutter Instruments Co.</td>
			<td>P-97</td>
		 </tr>
		 <tr>
			<td>Microforge</td>
			<td>Narishige Group</td>
			<td>MF-900</td>
		 </tr>
		 <tr>
			<td>Upright Patch clamp microscope</td>
			<td>Leica Microsystems</td>
			<td>DMLFSA</td>
		 </tr>
		 <tr>
			<td>Micromanipulator</td>
			<td>Siskiyou Inc.</td>
			<td>MX7500</td>
		 </tr>
		 <tr>
			<td>Digidata</td>
			<td>Molecular Devices</td>
			<td>1322A</td>
		 </tr>
		 <tr>
			<td>Amplifier</td>
			<td>Molecular Devices</td>
			<td>200B</td>
		 </tr>
		 <tr>
			<td>Acquisition software</td>
			<td>Molecular Devices</td>
			<td>pClamp9</td>
		 </tr>
		 <tr>
			<td>Axograph X</td>
			<td>Axograph</td>
			<td>Axograph X</td>
		 </tr>
		</tbody>
 </table>

patch clamp recordings from embryonic zebrafish mauthner cells

毛特纳单元（M-细胞）​​是设在硬骨鱼的后脑大reticulospinal神经元。他们参与被称为当机体感知的威胁发生了C-启动或逃跑反应特征行为的关键神经元。对M-细胞已被广泛研究在成年金鱼它已被证明收到了广泛的兴奋性，抑制性神经调节和信号1。我们一直在研究的M-细胞活性在斑马鱼胚胎为了研究突触发展的各个方面在脊椎动物制剂。在90年代末阿里和他的同事开发了膜片钳从M-细胞在斑马鱼胚胎，其中中枢神经系统基本完好2,3,4录制的准备。当时的目标是从后脑的神经元，脊髓神经元和躯干的骨骼肌，同时保持一个完整的脑脊髓内制备功能性突触的连接来记录突触活动。这种准备是在我们的实验室一直沿用到今天。要检查机制基本发育突触可塑性，我们记录兴奋性（AMPA和NMDA受体介导的）5,6和开发的M-细胞抑制性（GABA和甘氨酸）突触电流。重要的是，这种独特的准备可以让我们从准备返回相同的细胞（M细胞），以准备仔细研究突触可塑性和神经发育中的胚胎生物。通过该制备所提供的好处包括:1）完整的，功能性的突触连接到M-细胞，2）相对便宜制剂，3）大量供应现成的胚胎4）返回到相同的细胞类型的能力（ 即 M-细胞）的各项准备工作，以便在单个细胞水平突触发展，可以检查从鱼到鱼，5）由于胚胎的透明性质，整个筹备工作的成像。

在这个手稿中，我们从斑马鱼reticulospinal神经元的全细胞模式呈现的膜片钳记录的方法，特别是侧重于对M细胞。当然，也有可能在其他的膜片钳模式，如细胞贴附，由内向外和外列来记录。数据，人们可以得到的类型并不局限于我们在这里介绍的，但我们设法提供两个突触活动（兴奋和抑制）在电压钳模式，以及在电流钳动作电位的例子。作为有机体的年龄和M细胞培养更广泛和复杂的树突，以充分的电压钳和空间夹紧细胞的能力受到损害，并在电流钳模式变为所选择的记录。 图3显示了来自48个高倍视野胚胎在TTX的存在下得到的（1μM）和药理学试剂对兴奋性AMPA和NMDA受体电流的代表录音阻断GABA和甘氨酸受体。当M-细胞电压钳制在-60 mV时，突触电流是由于AMPA受体的激活（ 图3A）。采集与这些事件的分析允许一个记录的参数，如上升时间，下降时间，振幅和频率。 AMPA事件通常具有快的上升时间（〜0.1毫秒20-80％的上升时间）和衰减时间（约0.5毫秒），并显示出约25 pA的平均振幅。当M-细胞被钳位在40毫伏，所产生的外向电流是由于AMPA和NMDA受体的活性（ 图3C和3D）。 NMDA受体电流显示出比AMPA受体电流较长时间的课程，并且需要记录在正电位或镁的溶液来除去NMDA受体的镁块受体2,6。 图3D显示的平均mEPSC代表记录在+40 mV的混合AMPA和NMDA电流。 网络连接图4以显示记录从毛特纳细胞的mIPSC时有代表性的结果。发生在TTX的存在（1μM）和犬尿喹啉酸（1毫摩尔），以阻断AMPA和NMDA受体这些事件。这些事件表现出相当快的上升和衰减动力学由48个高倍视野和足够频繁收购其中不乏超过2-3分钟录音3， 图录作为注入去极化电流进入细胞的结果如图5所示动作电位。 M-细胞的48个高倍视野胚胎通常触发一个动作电位（ 图5B），尽管它们有时触发多个AP时与阈上刺激（ 图5A）注入。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td></td><td> 缩写 </td><td> 全名 </td></tr><tr><td> 1。 </td><td> MPSC </td><td>微型突触后电流</td></tr><tr><td> 2。 </td><td> mEPSC </td><td>微型兴奋性突触后电流</td></tr><tr><td> 2 </td><td> mIPSC </td><td>微突触后电流</td></tr><tr><td> 3。 </td><td> MΩ </td><td>兆欧姆（电阻单位; 10 6欧姆） </td></tr><tr><td> 4。 </td><td> GΩ </td><td>千兆欧姆（电阻单位; 10 9欧姆） </td></tr><tr><td> 4。 </td><td>毫渗量</td><td>毫渗透压（渗透压的单位; 10-3渗透压摩尔） </td></tr><tr><td> 4。 </td><td> TTX </td><td>河豚毒素</td></tr><tr><td> 5。 </td><td>三磷酸腺苷</td><td>三磷酸腺苷</td></tr><tr><td> 6。 </td><td> GTP </td><td>三磷酸鸟苷</td></tr><tr><td> 7。 </td><td>绿色荧光蛋白</td><td>绿色荧光蛋白</td></tr></tbody></table> 表1中。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td></td><td><strong&gt;设备名称</td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 1。 </td><td>解剖盘</td><td>华纳仪器</td><td> 64-0291 </td></tr><tr><td> 2。 </td><td> 0.001"钨丝</td><td>科学仪器服务公司</td><td> W406 </td></tr><tr><td> 2 </td><td>解剖显微镜</td><td>徕卡显微系统</td><td> MZ9.5 </td></tr><tr><td> 3。 </td><td>吸管拔轮器</td><td>萨特仪器有限公司</td><td> P-97 </td></tr><tr><td> 4。 </td><td> Microforge </td><td>成茂集团</td><td> MF-900 </td></tr><tr><td> 5。 </td><td>直立式膜片钳显微镜</td><td>徕卡显微系统</td><td> DMLFSA </td></tr><tr><td> 6。 </td><td>微操作机器人</td><td>锡斯基尤公司</td><td> MX7500 </td></tr><tr><td> 7。 </td><td> Digidata </td><td>分子器件</td><td> 1322A </td&gt; </tr><tr><td> 8。 </td><td>扩音器</td><td>分子器件</td><td> 200B </td></tr><tr><td> 9。 </td><td>采集软件</td><td>分子器件</td><td> pClamp9 </td></tr><tr><td> 10。 </td><td> Axograph X </td><td> Axograph </td><td> Axograph X </td></tr></tbody></table> 表2。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td></td><td> 解决方案名称 </td><td> 试剂和浓度（MM） </td></tr><tr><td> 1。 </td><td>解决方案剖析</td><td> 134氯化钠，氯化钾2.9，2.1的CaCl 2，1.2的MgCl 2，10 HEPES，10葡萄糖和0.02％三卡因</td></tr><tr><td> 2。 </td><td> MPSC细胞外液</td><td> 134氯化钠，氯化钾2.9，2.1 氯化钙 ，氯化镁1.2 2，10 HEPES，10葡萄糖和0.001 TTX </td></tr><tr><td> 3。 </td><td> MPSC细胞内的解决方案化</td><td> 134氯化铯，2 氯化镁 ，10 HEPES，EGTA 10，4的Na 2 +-ATP，0.4锂GTP </td></tr><tr><td> 4。 </td><td>动作电位细胞外液</td><td> 137氯化钠，氯化钾2.9，2.1 氯化钙 ，氯化镁1.2 2，10 HEPES和10葡萄糖（10μMD-筒箭毒碱） </td></tr><tr><td> 5。 </td><td>动作电位细胞内液</td><td> 130氯化钾，氯化钠2，10 EGTA，10 HEPES，4毫克，ATP和0.4锂GTP。 </td></tr></tbody></table> 表3。所有的解决方案外调整到290毫渗量和pH值7.8。所有细胞内的解决方案被调整为280毫渗量，pH值7.4。 <img alt="图1" src="/files/ftp_upload/50551/50551fig1.jpg" /> 图1。解剖盘和完整的后脑，脊髓准备。 （一）解剖和记录从WPI获得的菜。薄垫片仔细密封，涂于T他在培养皿的底部玻璃载玻片和的Sylgard被施加到井（B）48个高倍视野后脑-脊髓制备。后脑腹侧表面朝上和M细胞正好位于组织中的箭头的水平表面之下。 <img alt="图2" src="/files/ftp_upload/50551/50551fig2.jpg" /> 图2。 （A）利用Nomarski差分干涉对比孵化后不久从2旦的胚胎而获得的M-细胞的图像的图像记录自发兴奋性突触活动从M-细胞后取（B）该电池在充满荧光黄实验。来自12个经许可后改编。 <img alt="图3" src="/files/ftp_upload/50551/50551fig3.jpg" /> 图3。 （一）从M-细胞自发AMPA mEPSCs记录<html>在-60 mV的潜力。 （二）从记录中（A）中得到的平均mEPSCs（C）自发AMPA和NMDA（箭头）录得的M-细胞在+40 mV的潜在mEPSCs（D）从记录中获得平均mEPSCs （C）。谷氨酸mEPSCs记录于TTX的存在（1μM）以阻断动作电位，士的宁（5μM）和印防己毒素（100μM）以阻止甘氨酸和GABA电流。 <img alt="图4" src="/files/ftp_upload/50551/50551fig4.jpg" /> 图4。 （A）记录从一个M-细胞在-60 mV的潜在自发的mIPSC。 （二）从记录中（A）中得到的平均mEPSCs。的mIPSC记录于TTX的存在（1μM）以阻断动作电位，犬尿喹啉酸（1毫摩尔），以阻断谷氨酸受体活性和荷包牡丹碱（10μM）阻断GABA电流。 NT"FO:保持together.within页="总是"&gt; <img alt="图5" src="/files/ftp_upload/50551/50551fig5.jpg" /> 图5。动作电位记录从一个M-细胞，在该特定小区内的阈上刺激（0.48 NA）引起一些动作电位（A）中 ，但会刺激阈值仅导致在生产的单个动作电位（B）。 （C）电流协议中A和 B所示的痕迹。

Watch this Scientific Journal Video about Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells at JoVE.com

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

我们已经开发了一个完整的大脑，脊髓准备通过膜片钳记录从毛特纳神经元和其他reticulospinal细胞在斑马鱼胚胎，以记录和监测电活动。因此，我们能够记录兴奋性和抑制性突触电流，电压门控通道的活动和动作电位从胚胎发育的关键神经元。

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

patch-clamp-recordings-from-embryonic-zebrafish-mauthner-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.9K 次观看.  University of Alberta. 我们已经开发了一个完整的大脑，脊髓准备通过膜片钳记录从毛特纳神经元和其他reticulospinal细胞在斑马鱼胚胎，以记录和监测电活动。因此，我们能够记录兴奋性和抑制性突触电流，电压门控通道的活动和动作电位从胚胎发育的关键神经元。 

视频: Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

NRCC的膜片钳芯片上细胞的培养和电

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</subATP酶微囊的制备和表演膜片钳记录的Submitochondrial泡膜技术

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

从胚胎斑马鱼的毛特纳细胞膜片钳记录

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