We thank Joung Jang and Xin Guan for technical assistance. We thank Dr. Rory McQuiston of Virginia Commonwealth University for setting up our first patch clamp rig and advices on experimental procedures. We thank Dr. Samuel Wu for suggestions on voltage clamp recording. The work is supported by NIH grants EY013811, EY022228 and a vision core grant EY002520. C-KC is the Alice R. McPherson Retina Research Foundation Endowed Chair at the Baylor College of Medicine.

伦理批准 - 涉及受试动物的程序是按照规则和研究动物卫生准则的国家机构的规定进行的，所批准的机构动物护理和使用贝勒医学院的委员会。 1.外部和内部解决方案<ol><li>在视网膜剥离，并在随后电生理记录外液使用哺乳动物的林格氏液。制备哺乳动物林格氏上记录的一天从10倍原液（没有钙）溶液中，添加氯化钙2滴carbogenation的15分钟（95％O 2和5％CO 2）之后。最终1倍溶液含有（以mM计）:120氯化钠，5氯化钾，25的NaHCO 3，0.8 的 Na 2 HPO 4，0.1的NaH 2 PO 4，2的CaCl 2，1用MgSO 4和10 D-葡萄糖。 </li><li>使用两个内部解决方案，以表征SAC振荡。测试制备的溶液在长时间全细胞膜片钳记录对成年小鼠视网膜神经节细胞的有用性，并存储于500μl等份的验证批次在-20℃直到使用。 <ol><li>用于记录膜电位振荡，使用包含（以mM计）基于钾内部溶液:125 K-葡糖酸盐，8氯化钠，4的ATP的Mg，0.5立GTP，5 EGTA，10 HEPES和0.2％生物胞素（重量/ v）中。调节pH值至7.3 KOH。 </li><li>用于记录兴奋性和抑制性突触后电流，使用包含（以mM计）基于铯内部溶液:100铯甲磺酸盐，8氯化钠，4的ATP的Mg，0.5立GTP，5 EGTA，10 HEPES和0.2％生物胞素（重量/体积）。调节pH值至7.3用CsOH。 </li></ol></li></ol> 2.准备录制的日子<ol><li>制备具有开放孔的硝化纤维素膜，人工与来自钝为16G注射器针头制成的定制冲床冲膜和压平两个干净的玻璃板之间的薄膜。对于whole-安装视网膜，使中央孔直径为0.2mm以及由4较大的孔是1-1.2毫米直径围绕着它。 </li><li>使装载内部溶液到电极定制回填长丝，熔融10微升枪头在中间，直到它冷却变硬轻轻延长熔融部分。只是在使用前，用干净的刀片修剪细丝，直到它比补丁移液器稍长。 </li><li>在从硼硅玻璃管的可编程拉出器具有内部长丝拉补丁移液器。制造上实验当天的微量，并立即使用。 注:对于录音的SAC，我们用下面的车夫设置:热量:484;拉:0;速度:25;延迟时间:1;压力:400和462的硼硅酸盐管的外径和内径的斜坡值分别为1.65和1.0mm。移液器的电阻10-14MΩ。 </li><li>一个HR组织收获前，解冻被萨基内部解决方案的管纳克它为&gt; 30分钟的旋涡。 </li></ol> 3.视网膜解剖<ol><li>麻醉4％的异氟醚的动物中氧气直到动物失去反应到脚趾捏。颈椎脱位牺牲的动物。 </li><li>在1-2毫米从直指出虹膜剪视神经头离开切断视神经两个眼球。 </li><li>滚上干净的纸巾眼球，以清除血液，然后在carbogenated哺乳动物的林格氏液沉浸其中。按机器上使用23政针缘的孔，并伴随着微剪刀切缘平分眼球。删除使用细镊子角膜和晶状体。 <ol><li>对于视网膜整体安装，轻轻揭下用细镊子的色素上皮细胞的整个视网膜，并从向视神经乳头边缘4 1.5-2毫米正交削减。 </li></ol></li><li>沉浸穿孔硝酸纤维素膜，放入盘中，轻轻拖动与视网膜在它保利协鑫的一面朝上。制备整个安装视网膜当视神经乳头放入中心孔。与视网膜膜内转印到另一个干净的菜。轻轻压平视网膜用细刷油漆，看起来像一个马耳他十字打下了孔四边。 <ol><li>如果该视网膜的一小部分是在一个时间进行检查，切来自步骤3.3制备成3-4件用刀片与色素上皮保持连接每个眼罩。避光未使用的部分在carbogenated外部解决方案，12小时内使用。 </li></ol></li><li>吸干用干燥的滤纸的硝酸纤维素膜，并删除与镊子玻璃体和内界膜和漆刷。 </li><li>确保所有的视网膜边缘装配转印到记录室在底部密封的玻璃盖片之前完全附着到膜上。用真空脂膜固定到盖玻片和rehydratË与外部溶液视网膜。要小心，不要捕获任何气泡的组件下方。 </li><li>该室设置到一个直立式显微镜的阶段。以每分钟〜3毫升灌注速度用温水（34-35℃）carbogenated外液室。 </li><li>检查10倍物镜先下视网膜，然后用60倍的水浸泡镜头下看到微分干涉对比（DIC）和/或落射荧光GCL和INL神经元。为了形象化tdTomato表达的SAC，使用白光LED组合光源的波长554激发过滤器和581 nm的发射滤光片。 </li></ol> 4.全细胞膜片钳记录从平面贴装视网膜<ol><li>过滤通过针筒式滤器插入定制回填灯丝的内部溶液。 </li><li>插入灯丝成新鲜的拉微量和直到溶液覆盖银电极线为&gt;分配尖端附近的内部溶液为5mm。紧固微量到与抽吸杆的电极保持器，通过该电极内部的压力可以通过推动或拉动管10连接毫升的塑料注射器的柱塞进行调整。 </li><li>找到目标下的吸管，并把它归结为视网膜以上〜100微米。根据目前的跟随模式（I = 0），使用DC偏移为零立起的直流电压信号。通过吸移管注入固定振幅的方波电流测量电流钳（Ⅰ 钳 ）模式下的移液管电阻，而它是在浴中和通过转动r存取旋钮中和的差。使用对r存取旋钮读取由欧姆定律来计算吸液管电阻。 </li><li>慢慢把10微米视网膜上方的电极〜。施加正压到电极。观看枪头附近的反射变化，因为它接近视网膜。很快，但轻轻用力吸液管进入保利协鑫，降低了积极pressu立即重新。 </li><li>移动吸管向神经元标记。避免接触其他神经元，血管和Muller细胞endfeet。如果需要防止电极堵塞施加更大的正压力。 </li><li>直到一个凹坑是可见的标记的神经元的中线附近枪头定位。释放正压和允许质膜反弹到枪头。 </li><li>应用20〜120 pA的负电流，以移液管，以帮助一个千兆欧姆密封的形成。施加轻柔抽吸如果必要质膜拉入吸管。等待后密封形成5分钟以破裂细胞膜。这使得泻内部解决方案，通过灌流清除。用温和的吸力破裂膜。 </li><li>膜破裂后，而在电流钳模式，开启桥平衡，并使用r存取旋钮进行调整。 注:对于像国资委一小格，只需要稍作调整，如果有的话。 Alternatively，记录兴奋性和通过保持在氯化物的逆转电位的细胞（约-75毫伏）和谷氨酸（约0毫伏）分别在电压钳模式（V 钳位 ）抑制性突触后电流。检查并在必要时以适应偶尔的视网膜和/或显微漂移调整吸管的位置。 </li><li>到之前记录膜电位或电流变化，期间和药物治疗后，制备突触阻滞剂（ 如，CNQX，AP5，印防己毒素，筒箭毒碱等 ）和信道调制器（ 例如，氟吡汀，多巴胺，甲氯芬那酸等）新鲜上实验从冻结的股票一天。用稀释carbogenated林格氏液的药物。通过灌注以间歇方式应用它们。 </li><li>录制完成后，轻柔地从索玛吸管。从该室转移装配到一个干净的菜。分离和视网膜重新连接到另一个扁平硝酸纤维素膜上打孔不HOLES以固定期间保证视网膜平整度。 </li><li>通过在4％低聚甲醛浸渍在RT 30分钟固定的视网膜。取下硝酸纤维素膜在视网膜和用1×PBS广泛冲洗。由O / N染色用染料缀合的链霉在1×PBS稀释用0.4％的Triton X-100在4℃下14显示记录细胞的形态。在安装中防褪色视网膜和观察使用扫描共聚焦显微镜记录细胞。 注:涉及多聚甲醛，这是易挥发和有毒程序，应该在安全室草案进行。 </li></ol>

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The authors have nothing to disclose.

许多实验室已经从保利协鑫神经元记录台安装准备15-18，但我们的程序允许从INL神经元的记录。在此，我们强调的几个步骤是成功的日常记录的关键。 视网膜的新鲜度和平整度是有记录吸管穿透力很重要。在这方面，在视网膜的冲裁硝酸纤维素膜的牢固附着是非常重要的，并通过的溶液中，随后及时补液（步骤3.4-3.6）瞬态吸收实现的。在此短时间内，通常小于30秒，?...

As an easily accessible part of the central nervous system, the retina has for decades been a useful model in neuroscience studies. Genetic marking of neurons has allowed detailed characterization of synaptic connections in the retina. With many methodologies available to examine function and morphology of retinal neurons, the patch clamp recording technique has been instrumental in our current understanding of vertically transmitted signals in the retina. These signals are originated from photon absorption in photoreceptors and sent to brain visual centers through spiking of retinal ganglion cells (RGCs). Despite a large body of knowledge accumulated thus far, neural diversity in vascularized mammalian retina remains unsolved and obstructs the full appreciation of retinal circuits that subserve normal vision. This is in part because most recordings were performed on retinal slices to trade lateral circuit integrity for access to more proximal retinal neurons1-3. To gain a comprehensive picture on how retina computes visual signals, it is thus desirable to record neurons in flat-mounts wherein lateral connections, large and small, may be better preserved.
When synaptic transmission from photoreceptors to bipolar cells is interrupted due to a defective metabotropic glutamate receptor 6 (mGluR6) signaling pathway in depolarizing bipolar cells4-6 or simply as the result of photoreceptor loss in degenerated retinas7-10, many RGCs exhibit oscillatory activities. These oscillations originate from multiple sources, however the one involving gap junction coupling between AII amacrine cells (AII-ACs) and depolarizing cone bipolar cells (DCBCs) has received the most attention and hence is best understood1,7,11. We have found another source, which persists under pharmacological blockade of the aforementioned AII-AC/DCBC network and drives oscillation of OFF-type SACs in Rho&#916;CTA and Nob mice with deafferentated retinas7,8,12. Here we detail our protocol of preparing retinal flat-mounts for INL neuron recording. This approach uses commercial mouse lines (Jax stock no. 006410 and 007905) to mark cholinergic retinal neurons by fluorescent protein (tdTomato) expression that is identifiable under a fluorescent microscope equipped with contrast enhancing optics. Some experimental results acquired through this approach have been previously reported4,5,7,13.

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			<td height="42" style="height:42px;width:195px;">Fixed-stage fluorescent microscope with DIC</td>
			<td style="width:155px;">Olympus</td>
			<td style="width:219px;">BX51-WI</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Micromanipulators</td>
			<td style="width:155px;">Sutter</td>
			<td style="width:219px;">MP-225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Patch clamp amplifier</td>
			<td style="width:155px;">A-M System</td>
			<td style="width:219px;">AM2400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">AD converter</td>
			<td style="width:155px;">National Instrument</td>
			<td style="width:219px;">NI-USB-6221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Heater controller</td>
			<td style="width:155px;">Warner Instrument</td>
			<td style="width:219px;">TC-324B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Inline heater</td>
			<td style="width:155px;">Warner Instrument</td>
			<td style="width:219px;">SC-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Peristaltic pump</td>
			<td style="width:155px;">Rainin</td>
			<td style="width:219px;">Dynamax</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">pipette puller</td>
			<td style="width:155px;">Sutter Instrument</td>
			<td style="width:219px;">P-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Glass tube with filament</td>
			<td style="width:155px;">King Precision Glass</td>
			<td style="width:219px;">Customized</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Stimulator</td>
			<td style="width:155px;">A.M.P.I.</td>
			<td style="width:219px;">Master-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Biocytin</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">B4261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NaCl</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S6191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">KCl</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">P5405</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:219px;">BP328-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Na2HPO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NaH2PO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S5011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">C5670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgSO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">M1880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">D-glucose</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G6152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">K-gluconate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">ATP-Mg</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Li-GTP</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G5884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">EGTA</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">E0396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">HEPES</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">KOH</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Cs-methanesulfonate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">C1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CsOH</td>
			<td style="width:155px;">Sigma</td>
			<td align="right" style="width:219px;">232041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Syringer filter</td>
			<td style="width:155px;">Nalgene</td>
			<td align="right" style="width:219px;">171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">1 ml syring</td>
			<td style="width:155px;">Rainin</td>
			<td align="right" style="width:219px;">17013002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">10 ul pipette tip</td>
			<td style="width:155px;">Genesee Scientific</td>
			<td style="width:219px;">24-130RL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Streptavidin-488</td>
			<td style="width:155px;">ThermoFisher</td>
			<td style="width:219px;">S-11223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">10X PBS</td>
			<td style="width:155px;">Lonza</td>
			<td style="width:219px;">17-517Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina

哺乳动物视网膜是多种神经元类型构成的分层组织。为了理解如何视觉信号的错综复杂突触网络内进行处理，电生理记录经常用于研究单个神经元之间的连接。我们已经优化了遗传标记的神经元在这两个GCL（神经节细胞层）和INL鼠标视网膜的膜片钳记录（内核层）一台安装准备。在平面支架记录INL神经元是有利的过度分片，因为纵向和横向连接在前者配置保护，这样，具有大的横向分量的视网膜电路进行研究。我们已经用这种方法来比较视网膜镜像神经元结成伙伴的反应，如胆碱能爆无长突细胞（评估中心）。

ON-和从deafferentated小鼠视网膜关型的SAC的代表性记录示于图1。在这两个GCL和INL胆碱能细胞可通过tdTomato荧光可靠地识别和DIC下针对全细胞膜片钳记录（ 图1A）以揭示其膜电位（顶部的痕迹），并推动它（底部的痕迹， 图1B）突触电流的振荡。抑制和兴奋性突触电流通过保持在分别为0毫伏和-75毫伏，单元，电压钳的条件（ 图1B，<...

Watch this Scientific Journal Video about Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina at JoVE.com

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

此协议演示了如何从一台安装在准备视网膜神经元进行全细胞膜片钳记录。

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

patch-clamp-recording-starburst-amacrine-cells-flat-mount-preparation

Research

JoVE Journal

Neuroscience

8.1K 次观看.  Baylor College of Medicine. 此协议演示了如何从一台安装在准备视网膜神经元进行全细胞膜片钳记录。 

视频: Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

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

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 ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

全细胞膜片钳红外神经刺激机制的调查

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

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 ...

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. ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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 ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

成年小鼠视网膜电生理研究水平切片的制备

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...