这项工作是由研究资助，防盲和国家卫生部授予EY10542研究院。

1。视网膜切片准备<ol><li>组装所述腔室（ 图1中所示的设计）。将两个真空润滑脂，有孔玻璃珠间隔开〜8-10毫米，整个录音室，形成沟道灌流的和要在其中嵌入的视网膜切片。添加润滑脂几毫米更远超出这两个有孔玻璃珠的润滑脂作为堤坝和限制外溢的第二胎圈。将一个小的三角片KimWipe上面的腔室的端部，以确保流体接触的参比电极。 </li><li>按一块硝酸纤维素膜（〜5×10毫米，0.8微米毛孔）平成两个小珠真空油脂对显微镜载玻片。避免将油脂直接的中心下的硝酸纤维素膜，因为这样会导致视网膜附着。 </li><li>要准备组织切片机，打破了双边缘刀片成4块，并附加一个切片臂。切薄片nitrocellul（作家）膜，以确保切削刃的刀片平放在对录音室，因此，通过硝酸纤维素膜干净地切割。 </li><li>两栖类生理盐水溶液（ 表1）在解剖站上冰保持小烧杯中。 </li><li>安乐死蝾螈斩首。 Hemisect头了得sagitally通过脊髓髓。顶上的油毡块与两栖动物盐水浸湿的棉片将一半的头。头部的另一半可以，用潮湿的纸巾包裹，并储存在4℃下，使用在当天晚些时候。 </li><li>的眼睛Enucleate。使用小维纳斯剪刀，切开皮肤，眼睛周围的轨道连接。释放前面的眼睛周围的轨道组织后，扯人眼球向前滑动的剪刀划破的眼部肌肉和视神经下眼，使眼睛的轨道。 </li><li>将去核的眼睛在床上棉花油毡块。丢弃的半头。修剪多余的眶隔脂肪，从后面的眼睛。 </li><li>在角膜的中心做一个小切口，用锋利的手术刀片。取出角膜切口延长径向切向锯齿缘滑动精细维纳斯剪。周围切锯齿缘通过旋转油毡块或间棉削减。 </li><li>切割后眼睛周围的所有的方式，去除角膜和晶状体拉出侧面眼罩。到坚硬的表面移动产生的眼罩油毡块蘸与两栖动物生理盐水。它切成三分之二用锋利的刀片，用细锯议案，以确保您已经削减了所有的方式，通过巩膜。 </li><li>将一份或两份接目环件上的硝酸纤维素膜与视网膜表面朝下。额外盐水淹没残片，并将其放置在冰箱〜4℃ </li><li>戈ntly按一块眼罩对硝酸纤维素膜用细镊子。淹没硝酸纤维素膜和眼罩片的边缘与KimWipe帮助视网膜坚持几滴冷两栖动物盐水和印迹。再次浸在眼罩和硝酸纤维素膜与两栖类冷生理盐水溶液剥离巩膜/脉络膜/视网膜色素上皮细胞的分离的视网膜（它可以出现粉红色的，由于存在未漂白的视紫红质）数滴。如果有必要，切断视神经，视网膜释放。 </li><li>如果视网膜没有遵守得紧紧的，排走生理盐水与KimWipe拉扯视网膜，更坚定地走下去，到硝酸纤维素膜。更换生理盐水。重复，如果有必要的话。 </li><li>冷两栖动物生理盐水填充室转移到组织切片机的阶段。视网膜和硝酸纤维素膜切片切成细条，从一端到另一工作通过转动千分尺在增量为125微米。按刀片轻轻地但坚定地通过视网膜和硝酸纤维素膜。 </li><li>视网膜切片转移硝酸纤维素膜条移动录音室的主渠道。提起带状免费膜，然后拿着它到位室下方移动时，一定要保持片淹没。添加的硝酸纤维素膜在真空润滑脂条的边缘，旋转90度，以查看在视网膜层。 </li><li>按平贴在玻璃表面的硝酸纤维素膜。即使有，不是对每一个作品的视网膜，将带以规则的间隔（〜1毫米）沿其整个长度的灌注通道，帮助打破的表面张力和改善流体流动的硝酸纤维素膜。 </li></ol> 2。配对的全细胞记录<ol><li>所有切片后已经转移，移动录音室，一个堂堂正正的阶段，固定舞台显微镜和附加参考电极引线。重点在片使用长工作距离，开水浸泡，40-60X的目标。显微镜应放置在空气表来抑制振动，封闭在一个法拉第笼，以减少电子干扰。 </li><li> Superfuse连续切片的速率为1毫升/分钟，两栖类盐溶液与100％O 2鼓泡。连接吸气，确保流入和流出是平衡的。流出可调节，旋转吸入针的斜角端，或通过移动的KimWipe在该腔室的端部从流出针靠近或远离。该制剂可以保持在室温或冷却用珀耳帖（Peltier）器件，或通过简单的设置显微镜载物台的一个冰袋。 </li><li>切片检查变暗或红外光下找出一对感光细胞 - （棒或圆锥）和附近的水平或双极细胞 - 全细胞记录的目标。棒可我dentified大胞体和突出的棒状外段（ 图2A）。视锥细胞是小于棒，有小的锥形外段。双极细胞，水平细胞胞体中的细胞体在内核层（INL， 图2B和2C）的最外排。 </li><li>在准备切片，用移液器拉马制造微量硼硅玻璃（1.2毫米外径，0.95毫米内径与玻璃纤维）。每个微管的顶端应为〜1-2微米的直径。 </li><li>使用非金属填充针（ 例如，一个从1毫升注射器或微滤制造），填补移液管与细胞内的溶液（ 表1），和连接到所述电极保持器。 </li><li>显微镜物镜略有提升。物镜下方的感光体移液管的位置，然后下降，使针尖位于正上方的片。重复与t他第二次吸管。 </li><li>调整放大器的基准电流电平在任何偏移。检查吸液管电阻5-10 mV的去极化脉冲。我们通常使用的移液管，范围从10至15MΩ的长锥形轴和低渗透压的两栖动物液的结果。具有较高的摩尔渗透压浓度的哺乳动物的解决方案中，这些相同的移液管表现出〜8-12MΩ的电阻值。虽然我们已经使用了尖端直径较大的电阻值3-4MΩ两栖动物解决方案，提供一个较低的访问性的优点抵消在细胞膜上的密封难度更大和更快速的破败的钙电流和其他第二信使敏感的反应。 </li><li>轻微的正压力，位置，同时施加在突触后的移液管，使接触水平或双极性电池主体的。如果是，定位的突触前的移液管，以便接触一个棒或锥照相感光体的细胞体。录音APPEAR更稳定枪头接触时，尤其是在锥内段，而不是苏摩。 </li><li>监测的阻力，松开正压后突触吸管上。有时，正压力的释放是足以形成密封千兆欧姆。如果没有，申请轻柔吸用1毫升注射器或经口。尖阻力后已经成长为&gt; 100MΩ，适用于保持电位为-60 mV。获得千兆欧姆密封后，空出的任何吸管的电容瞬变和感光吸管重复密封的过程，将保持电位为-70 mV。 </li><li>用你的嘴或注射器吸每个单元又破裂的补丁。棒，锥细胞和双极细胞通常会破裂，轻轻吸。获得全细胞配置水平细胞，可能需要更大的吸力（ 即用3毫升注射器）的组合具有较强的快速电压脉冲“ZAP”F膜片钳放大器eature。破裂的膜和建立全细胞配置将是显而易见的外观全细胞电容瞬变。 </li><li>确认身份突触后细胞生理施加一道光闪过，并提供了一系列的电压从-120至+40 mV在20 mV递增（ 图3和图4）步骤。 </li><li>要评估如果对细胞的突触连接，提供一个简短的（25-100毫秒），60 mV的步去极化的光感受器（到-10 mV，L-型电压门控钙电流峰值附近），并期待二阶神经元突触后电流（ 图5）。一个强大的去极化步骤应该唤起快，瞬态向内突触后电流在突触后水平或OFF双极细胞引起一阵囊泡释放锥（ 图5）。 </li></ol>

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没有利益冲突的声明。

视网膜切片准备已经被证明是非常有用的分析视网膜处理视觉信息的电路和机制。同时获得全细胞记录前和突触后神经元的能力一直在这一努力中特别有用。配对的全细胞记录片更容易实现比平面式视网膜制剂，因为不同的视网膜层暴露。此外，由于其大的视网膜神经细胞，蝾螈有悠久的历史，作为视网膜的准备，因此提供了一个特别良好的特点的模型系统。 通过实践，蝾螈视网膜的健康片可以定期编制。几个关键的步骤，可以使成功和失败之间的差异。 1）确保刀片安装的组织切片机上，以便它贴在玻璃表面和片平放在干净，但组织和underlyi毫微克的硝酸纤维素膜。通过硝酸纤维素膜，如果你已经做了一个干净的切割，你应该听到一声微弱的点击刀片罢工载玻片表面。 2）确保视网膜坚持到硝酸纤维素膜。否则，视网膜可以浮动相差的膜，在任何步骤中的程序。 3）不要切割片暴露在空气中，因为这将损害许多表层细胞。 4）确保片和硝酸纤维素膜对玻璃滑动平躺，使视网膜层解剖显微镜下是显而易见的。 5）平衡灌流流入和流出率为了避免溢出录音室。这可以防止在溶液水平的突然变化，这会导致突然的组织的活动。 6）选择健康的细胞对彼此接近。光滑胞浆细胞比健康细胞胞浆呈颗粒状。更深切片的细胞​​更可能保留完整的突触CON触点。 7）确保枪头不破或其他组织或碎片的道路上蹭着细胞。 8）检查，以确保它没有被阻塞碎片或泡沫，这两者都可以使它很难获得高质量的全细胞记录吸液管电阻。 ，而不是视网膜附着到硝酸纤维素滤纸上，一些调查嵌入视网膜在一个块中的琼脂，并使用一个vibratome切割视网膜切片获得。虽然我们还没有试过这种方法，11 Kim 等人讨论这两种方法的优点。根据他们的经验，琼脂为基础的方法提供了更一致的平片产量与划定视网膜层，但滤纸为基础的方法产生健康的感光细胞。 杆和视锥细胞负责传导光线进入的膜电位变化。杆和视锥细胞的膜电位与配对录音，可以manipul直接生成，因此产生光的反应能力，同时有助于确定细胞类型，可能是必不可少的。因此，我们经常准备切片在白光。然而，即使在明亮的光照下准备，蝾螈视网膜神经细胞可以产生大量的光反应所示，由图中的响应。 3。这部分是由于到生色团外段大体积比较大的水库，但也可能反映Müller细胞的再生能力的11 -顺式视网膜视锥细胞12。要获得充分的暗适应光的反应，我们可以准备切片在红外线照射下。解剖红外光下，我们重视GenIII图像增强（Nitemate NAV3，利顿工业公司，亚利桑那州坦佩）解剖显微镜的目镜和一个红外LED手电筒照亮的组织。对于切片和其他程序都没有进行解剖显微镜下，我们聘请了头戴式IMAGE增强。安置的补丁移液器，切片使用红外敏感CCD相机（WATEC公司， 如 WATEC 502H米德尔敦，NY）安装到直立，固定阶段显微镜可视化。这些预防措施，可以得到单光子灵敏度6，13杆响应参展。 在视网膜切片工作的一个限制是长期大视野视网膜神经元细胞过程可能会失去他们的许多树突在切片过程。视网膜切片的准备，因此更有益的研究的生理的细胞的突触接触涉及靠近细胞体的过程。两栖类和哺乳动物的视网膜共享许多相同的细胞类型，并利用类似的生理机制14-16。蝾螈视网膜的良好模型的哺乳动物的视网膜中的许多方面的一个重要的区别似乎是一个专用杆通路的存在下在哺乳动物INVOlves专门杆双极细胞接触到AII无长突细胞14。蝾螈视网膜的另一个局限性是国内少数专门为这个物种的遗传工具。然而，抗体和shRNA试剂，目标以及在其它物种中保守的区域可以成功地用于在蝾螈，以及许多小分子抑制剂和肽试剂。此外，随着技术的一些修改，视网膜切片可以准备一些这些工具更容易获得来自其他物种。 除了配对的全细胞记录其效用，蝾螈视网膜切片准备也适合各种其他方法。正如上面所讨论，视网膜切片可用于研究与各种电压钳位协议17的组合的光反应。视网膜神经细胞也可以被载入敏感的荧光染料的Ca 2 +，Cl -的，或Na+引入通过的补丁吸管或15,18-20浴应用。束缚的突触带21上的荧光肽可以通过引入补丁吸管和用于成像的发带10，或荧光素共轭时，急性和选择性地损坏条带22。我们也使用与量子点结合的视网膜切片监测运动杆和锥突触终端23的单独的钙离子通道。因此，垂直视网膜切片是一种多用途的研究基础突触机制和独特的处理功能，在视觉信号传导通路的第一突触进行实验准备。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/material</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Tissue slicer</td>
 <td>Stoelting</td>
 <td>51425</td>
 <td></td>
 </tr>
 <tr>
 <td>Double edge razor blades</td>
 <td>Ted Pella, Inc</td>
 <td>121-6</td>
 <td></td>
 </tr>
 <tr>
 <td>Nitrocellulose membranes</td>
 <td>Millipore</td>
 <td>AAWP02500</td>
 <td>Type AAWP 0.8 mm pore</td>
 </tr>
 <tr>
 <td>Borosilicate glass pipettes</td>
 <td>World Precision Instruments</td>
 <td>TW120F-4</td>
 <td>1.2mm OD 0.95 mm ID</td>
 </tr>
 <tr>
 <td>Ag/AgCl pellet</td>
 <td>Warner</td>
 <td>E206</td>
 <td></td>
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 <tr>
 <td>MicroFil</td>
 <td>World Precision Instruments</td>
 <td>MF34G-5</td>
 <td>34 ga. Filling needle, 67 mm long</td>
 </tr>
 </tbody>
</table>

simultaneous whole-cell recordings from photoreceptors and second-order neurons in an amphibian retinal slice preparation

在视网膜神经科学的中心任务之一是了解视网膜神经细胞的电路，这些连接是如何塑造的信号传递给大脑负责。光子被检测到的视网膜杆和锥光感受器，它的能量转换成一个电信号，传送到其他的视网膜神经细胞，在那里进行处理和通信中心在大脑中的目标，通过视神经。重要的早期洞察视网膜电路和视觉处理来自卡哈尔1,2的组织学研究，后来，从电生理记录扣球视网膜神经节细胞的活性-输出的视网膜细胞3,4。 在视网膜中的可视处理的详细了解，需要了解的信令在视网膜神经节细胞的途径从感光体中的每个步骤。然而，许多视网膜细胞类型是牙钻IED在组织深处，因此相对人迹罕至的电生理记录。这种限制是可以克服工作与垂直切片，其中居住于视网膜层细胞清晰可见，电生理记录访问。 这里，我们描述了一种用于从仔鱼，：老虎蝾螈（tigrinum钝口螈属 ）的垂直剖面的视网膜。虽然这最初是为准备录音用锋利的微电极5,6双全细胞电压钳记录，我们描述的方法，从感光细胞和二阶的水平细胞和双极细胞中，我们处理的感光膜电位，同时记录后在水平或双极细胞的突触反应。虎蝾螈的光感受器是远远大于那些哺乳动物物种，这是一个理想的制剂，其中承接吨他在技术上具有挑战性的实验方法。这些实验着眼探测突触带信号性能描述-一个专门的突触结构，发现在一个只有极少数的神经元，包括杆和视锥细胞，非常适合保持率很高的滋补神经递质的释放7 ，8 -和它有助于视网膜突触这首独特的信令性能。

代表垂直切片蝾螈视网膜神经元对光反应的痕迹， 如图3所示。锥，水平细胞，双极细胞和OFF显示外向电流响应发病轻。突出内向电流水平和双极细胞记录后，灯闪光偏移，因为他们去极化的光感受器引起的谷氨酸释放增加。双极细胞响应内向电流在轻发病因符号反相的代谢型谷氨酸受体信号级联和激活的TRPM1频道9。通过IV的关系（ 图4），水平细胞，双极细胞，可以彼此区分。水平细胞通常具有的直链或四内向整流和低输入电阻（&lt;500MΩ; 图4A），而双极细胞具有高输入电阻（0.5-2GΩ）向外整流I - V（ 图4B）。图5显示了有代表性的结果从一个锥形水平细胞对锥关对双极细胞（ 图5B）（图5A）的录音。在每个从保持电位为-70 mV，去极化到-10 mV锥引起了电压门控钙电流水平或双极细胞锥体和快速向内EPSC。这种强烈的刺激足以清空容易释放池，导致EPSC〜47 PA /色带10〜20小泡从每个突触带。 图5A水平细胞EPSC 232 PA，表明它收到5带状触点从突触前锥。类似的估计从178 PA工作小组在关闭双极细胞（ 图5B），表明它收到了4个带状触点从突触前锥。 <table border="1"><tbody><tr><td></td><td><s阮富仲&gt;两栖动物生理盐水</td><td> 突触前吸管 </td><td> 突触后吸管 </td></tr><tr><td>氯化钠</td><td> 116毫米</td><td> 3.5毫米</td><td> 3.5毫米</td></tr><tr><td>氯化钾</td><td> 2.5毫米</td><td></td><td></td></tr><tr><td> 氯化钙 </td><td> 1.8毫</td><td> 1毫米</td><td> 1毫米</td></tr><tr><td> 氯化镁 </td><td> 0.5毫米</td><td> 1毫米</td><td> 1毫米</td></tr><tr><td> HEPES </td><td> 10mM的</td><td> 10mM的</td><td> 10mM的</td></tr><tr><td>葡萄糖</td><td> 5毫米</td><td></td><td></td></tr><tr><td>铯-谷氨酸* </td><td></td><td> 40毫米</td><td></td></tr><tr><td>葡萄糖酸铯** </td><td></td><td> 50毫米</td><td> 90毫米</td></tr><tr><td>四乙基氯化铵</td><td></td><td> 10mM的</td><td> 10mM的</td></tr><tŗ&gt; <td> ATP-Mg系</td><td></td><td> 9毫米</td><td> 9毫米</td></tr><tr><td> GTP </td><td></td><td> 0.5毫米</td><td> 0.5毫米</td></tr><tr><td> EGTA </td><td></td><td> 5毫米</td><td> 5毫米</td></tr><tr><td> pH值*** </td><td> 7.8 </td><td> 7.2 </td><td> 7.2 </td></tr><tr><td>渗透压**** </td><td> 245毫渗量</td><td> 240毫渗量</td><td> 240 MOSM </td></tr></tbody></table> *铯-谷氨酸是由用40mM的CsOH电极内液中的40mM的L-谷氨酸。 ** 1 M库存CS-葡萄糖酸是由D-葡萄糖酸45-50％的CsOH中的解决方案。 ***用NaOH调节pH值应为细胞外液和吸液管的解决方案的CsOH。 ****保持略低于液的渗透压，细胞外液，防止细胞肿胀和提高长寿的记录。 表1。细胞内和细胞外的标准解决方案，此协议中使用的组件和参数。 <img alt="图1" fo:content-width="3.5in" fo:src="/files/ftp_upload/50007/50007fig1highres.jpg" src="/files/ftp_upload/50007/50007fig1.jpg" /> 图1。录音室设计（AC）顶部，侧面和录音室的显示尺寸的剖视图。加工室从2毫米厚的丙烯酸（D）组装室。灌流通过聚四氟乙烯管（10厘米长的流入管 ; 24LW型）进入燃烧室。灌流除去施加轻度抽吸到斜面20的另一侧的腔室的尺寸的金属管。此输出管的相邻的是一个颗粒参比电极Ag / AgCl电极。此参比电极的导线被连接到参考输入的探头。一个小三角KimWipe的被放置在参比电极，以保持其与溶液接触，并调节溶液流进流出管。成的腔室的凹入边缘放置的玻璃显微镜载片上形成的腔室的底部。该滑动固定在适当位置，用珠真空润滑脂。条状的硝酸纤维素膜与视网膜片嵌入在真空润滑脂的有孔玻璃珠形成沟道的溶液流。使片之前，一个5×10毫米长的硝酸纤维素膜固定到所述腔室与两个小珠真空润滑脂。眼罩被放置一块玻璃体侧倒在这硝酸纤维素膜一旦视网膜坚持解除了。 <a href="https://www.jove.com/files/ftp_upload/50007/50007fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/50007/50007fig2.jpg" /> 图2。染料填充细胞对已经编制rtical片A）杆水平细胞和突触耦合充满了对比的荧光染料，通过补丁吸管在同时全细胞记录的图像。路西法黄色（2毫克/毫升）被列入杆液（黄色）和sulfarhodamine B（1毫克/毫升），水平细胞吸管溶液（紫色）被列入。使用旋转圆盘共聚焦显微镜（Perkin Elmer公司制Ultraview LCI）配有一个冷却的CCD相机（奥卡ER），并安装到固定载物台的显微镜（尼康E600 FN与60X，1.0不适用水浸物镜）捕获荧光图像。这些图像是亮场图像叠加在相应的视网膜片使用Adobe Photoshop B）图像的圆锥OFF双极细胞和突触耦合。双极细胞的轴突终末分支内核层边界附近的内网状层的最外层（S1）板下。 <strong&gt; C）的锥水平细胞和突触耦合的图片。需要注意的是锥形端子显着大于杆轴索终端。虽然水平细胞可以被识别的长椭圆形的形状，它们的细胞体，细胞体的ON和OFF型双极细胞的INL不能很容易地记录之前区分。然而，我们可以OFF双极细胞的细胞体在光学网络中，可以区分由内层和外层部分的情况下从锥锥驱动目标位移。标尺为20微米。 <img alt="图3" fo:content-width="2.5in" fo:src="/files/ftp_upload/50007/50007fig3highres.jpg" src="/files/ftp_upload/50007/50007fig3.jpg" /> 图3。光诱发锥（A），水平细胞电流电压钳记录下从四个不同的视网膜神经元在500毫秒明亮的白光一闪。 <stronG&gt;（C）（B），双极细胞和关闭所有回应光外向钾电流（D）内向电流双极细胞的相同的光刺激。关闭双极细胞（C）基线噪声展出，反映了持续释放时，减少光感受器超极化光在黑暗中的突触小泡。从这四个细胞中获得了单独录音，使用切片准备在白色灯光下的响应。在这些实施例中使用的白色光刺激的强度产生响应，相当于1×10 5个光子/秒个/μm2的580nm的光子通量的水平和双极细胞。 <img alt="图4" fo:content-width="3in" fo:src="/files/ftp_upload/50007/50007fig4highres.jpg" src="/files/ftp_upload/50007/50007fig4.jpg" /> 图4。电流-电压（IV）为h的关系orizontal和关断双极细胞（A） 上方的面板中 ，诱发的一系列的150毫秒的电压阶跃20 mV递增（ 底部面板 ），水平细胞应用从-120到+40 mV的膜电流。水平细胞通常具有低的输入电阻和直链或内向整流型IV的关系，（B）的 IV关系，响应一系列的电压步骤断开双极细胞。双极细胞具有更高的输入电阻和一个向外整流的四 ，由于激活电压门控钾电流。 <img alt="图5" fo:content-width="2.75in" fo:src="/files/ftp_upload/50007/50007fig5highres.jpg" src="/files/ftp_upload/50007/50007fig5.jpg" /> 图5。 锥锥电压门控性钙电流（A）记录（我CA 配对记录数据的例子。 </sUB），响应于一个100毫秒的步骤从-70毫伏（ 锥V M），保持电位为-10 mV。泄漏和电容瞬变减去使用P / 8泄漏减法协议。甲快兴奋性突触后电流（EPSC; HC PSC），同时记录，可从水平细胞，表明这两种细胞的突触耦合（B）的工作小组，在另一个同样的刺激响应的记录，可在关断双极细胞锥。

Watch this Scientific Journal Video about Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation at JoVE.com

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

我们描述了制备薄视网膜片从水产虎蝾螈（<em&gt; Ambystoma tigrinum</em&gt;），并解释我们如何使用这些切片研究获得双全细胞电压钳记录，从感光细胞和二阶的水平细胞和双极细胞在视网膜突触处理。

同时光感受器和二阶神经元在两栖类动物的视网膜切片制备全细胞记录

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

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JoVE Journal

Neuroscience

13.6K 次观看.  University of Nebraska Medical Center. 我们描述了制备薄视网膜片从水产虎蝾螈（ Ambystoma tigrinum），并解释我们如何使用这些切片研究获得双全细胞电压钳记录，从感光细胞和二阶的水平细胞和双极细胞在视网膜突触处理。

视频: Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

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

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

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 ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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 ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...