我们要感谢吉拉德西尔伯贝格，米歇尔皮尼亚泰利，托马斯·K.伯杰，卢卡Gambazzi，和索尼娅·加西亚就改善了膜片钳过程自动化宝贵意见。我们感谢Rajnish兰詹的宝贵意见，并与软件实施援助。这项工作是由欧盟Synapse项目和部分由人类前沿科学计划提供部分资金。

1。设备准备<ol><li> 从电脑控制机械手 <ol><li>通过串口（RS-232）每个微操作控制盒连接到电脑。 </li><li>实施命令进行定位，查询和调整设置通过串口发送。考虑到速度和硬件兼容性问题的C / C + +被推荐为编程语言。 </li><li>标准化的操纵器的基准系统，以使零是相对于所述电动机和积极运动从马达引导离开最接近的可能位置。 </li><li>定位显微镜在其中央位置2毫米以上的标本焦平面（坐标[0，0，2000]）。 </li><li>存储，每个操纵器，该局部坐标系，使在视显微镜领域的中心到被观察的移液管的尖端。这是对每个电极的初始参考点。 </li></ol></li><li> Visua丽泽电极的位置。能够在实验中要跟踪的每个电极的位置是非常有用的。图形表示是解决这个问题的最直观的方法。为此各电极和参考的系统，在显微镜的必须匹配。做到这一点的简单方法是: <ol><li>把一个电极的前端到所述视场的中心。 </li><li>存储在机械手的各轴的位置和显微镜的各轴。 </li><li>执行与该机械手相对大的运动（1毫米）的x轴。 </li><li>找到一次只移动显微镜的尖端。 </li><li>计算在显微镜位置的差异，因为它是最后一次测量。这些都是在电极轴运动到显微镜轴上的投影。 </li><li>为Y和电极操纵器的Z轴上2.5 - 重复步骤2.3。这使得投影矩阵到显微镜轴来确定（&lt;STRONG&gt;图2），也称为余弦矩阵: <img alt="式（1）" src="/files/ftp_upload/50630/50630eq1.jpg" /></li><li>反转这个矩阵，使其能够计算运动，在三维空间中，以达到在显微镜坐标系统中的给定位置所必需的电极操纵器: <img alt="公式2" src="/files/ftp_upload/50630/50630eq2.jpg" /></li><li>使用初始基准点和余弦矩阵确定在显微镜各电极的位置坐标。 </li><li>显示图形的基础上，在显微镜坐标，每个电极的定期的位置。我们选择使用的C / C + +的绘图库（GDI +）来绘制电极位置每隔40毫秒。 </li><li>重复步骤1.2.1 - 1.2.7每个机械手的角度被改变，或者如果需要几毫米的位置变化时，例如，当电极的类型而改变。 </li></ol></li><li> 使存储positio的相关特征纳秒的组织，如细胞或解剖参考点，在显微镜坐标。 </li><li> 获取视频和覆盖相关的信息。 <ol><li>机械地对准显微镜的运动的x-轴与显微镜照相机的水平轴。 </li><li>在计算机上安装有视频直播和覆盖能力和软件开发工具包（SDK）图像采集卡。 </li><li>实施实时视频显示操作的SDK。 </li><li>转换显微镜的坐标系统到摄像机参考系统通过适当的平移和缩放。 </li><li>绘制有关的功能中的实时视频所产生的图像以大约40毫秒（ 图3）定期照相机坐标和覆盖。 </li></ol></li><li> 控制放大器。 <ol><li>使用放大器的软件来控制从接口放大器设置。 </li></ol></li><li> 合作ntrol示波器。 <ol><li>使用串行端口的示波器连接到PC。 </li><li>确定示波器规模，耦合和时间分辨率在电压钳的不同步骤的膜片钳程序（ 如电极的浴室，封口平整，全细胞配置）和电流钳。 </li><li>发送相应的示波器设置命令时放大器的命令是从接口发出。 </li></ol></li><li> 控制吸管的压力。 <ol><li>根据图4的组合的压力控制系统。 <ol><li>使用12 V / 5 V电源适当地供给各电子元件。 </li><li>一膜泵的输出连接到正压缓冲液（100毫升的容器）。 </li><li>一个膜泵的输入端连接到负压缓冲液（100毫升容器）。 </li><li>移液器支架管连接在pressu压力传感器重新控制系统和气动阀连接到主压力舱。 </li><li>各压力缓冲器的连接连接到主压力室的阀。 </li><li>连接主压力室与大气之间的阀。 </li><li>压力传感器连接到主压力舱。 </li><li>压力传感器连接到每个缓冲区。 </li><li>压力控制系统连接到数据采集板。 </li><li>每个压力传感器连接到一个模拟输入。 </li><li>每个阀门连接到数字输出。 </li></ol></li><li>去除大气压力打开所有阀门除了那些连接到隔膜泵和减去测得的压力来自所有传感器的偏移。 </li><li>实现压力控制<ol><li>在接口定义控件激活或去激活每个移液器正压控制。 </li><li>定义吸管最小正压的S约70毫巴。 </li><li>定期（每0.5秒）检测时主动下压控制在吸液管压力低于设定的阈值。 </li><li>当阈值交叉打开正压缓冲到主压舱和车厢这对有问题的吸管在短暂的周期（20毫秒），直到吸液管压力高于阈值。关闭所有其它阀门。 </li><li>去激活更多的压力控制，对目标细胞的最终办法启动。 </li></ol></li><li>应用负压密封形成<ol><li>关闭所有阀门，并打开向主压舱和车厢这对吸管有问题的实验者需要通过保持按下按钮期间负压缓冲阀。 </li></ol></li></ol></li><li> 命令集中到一个人机接口设备。 <ol><li>连接市售wireleSS手柄到PC。 </li><li>实施操纵杆状态读数。例如，使用DirectX库的C / C + +来进行读数，每5毫秒。 </li><li>与以前的状态比较当前状态，以检测哪些按钮已被按下，释放或保持按下自最后一次一步。 </li><li>在手柄的功能分配到每个按钮。这种映射的一个例子示于图5。 </li></ol></li></ol> 2。膜片钳程序<ol><li> 制备所关注的区域的脑切片。 </li><li> 放置的兴趣的脑切片，用在显微镜的位移范围的中心感兴趣区域。 </li><li> 小区选择 <ol><li>通过浏览与显微镜识别感兴趣的细胞。存储单元的基础上，显微镜坐标系的位置与右上关注单元的顶部的实况视频显示器上的鼠标点击。 </li&gt; <li>图形化界面将显示选定的单元格，以及将微量的全球概览和软件将添加一个标记上，将要在图像上叠加的单元格位置。此外，该单元的图像在文件&#39;细胞＃。JPG"捕获以供将来参考。 </li></ol></li><li> 细胞的归属地移液器 <ol><li>选择感兴趣的细胞后，分配哪些吸管将记录每一个细胞。图形化界面提供了应设置的分配控制。通过选择复选框"显示最终位置"可视化的最终配置的预览。 </li><li>选择它的最终位置预览期望或检查的"全选"复选框，每个吸管。如果需要禁用当前位置显示为更好的显示效果。 </li></ol></li><li> 准备移液器 <ol><li>填写移液器（6 - 8MΩ通常是最好的，如果很多移液器使用）无线TH细胞内的解决方案并将它们加载到其持有人。放置的探头在他们的注视，但不滑动他们前进，避免接触浴枪头。 </li><li>使正压控制和选择所有移液管，以确保提示将保持清洁。轻轻滑入到位每个探头。 </li></ol></li><li> 定位移液器吸头 <ol><li>显微镜定位与聚焦3毫米薄片按按钮R2固定按钮"A"，而上面的中心位置。使用相应的位置上的每个机械手按下按钮，L2为存储从以前的实验中同时按住按钮A。 注:在这一点上应该有一个吸管鉴于无论是独特的阴影或沿吸管的轴的小运动应该足以观察它在大多数情况下。补偿在吸管的形状略有不同，必须手动执行。 </li><li>把枪头成焦点。将吸头在视频显示的红色圆点中央，而无需移动显微镜（它应该仍然是在位置[0，0，2000]）。 </li><li>通知移液器是在显示器的中心按下按钮z按住按钮C之后每个移液器吸头所在的软件，发送吸管向后，使下面的人能够同时按住按钮A位于按L1键</li></ol></li><li> 走近细胞 <ol><li>一旦所有的枪头已精确定位，每个吸管被归结为一个单元格，自动定位接近各自细胞的移液管。只需右键单击该组细胞的中心，并选择弹出菜单上的选项&#39;集群&#39;。上会出现，选择所有你想要在这个时候定位移液器，点击&#39;去了所有检查移液器&#39;群集选项窗口。重复此操作所关注的细胞的每个集群。 </li><li> PE手动rform最后的办法。相对于细胞的吸移管的位置可以被指定不同的，但通常是简单地200μm的距离的移液管和200μm的上方在垂直方向上，这是足以使移液管的尖端的组织以外的轴的小区。等到移液器的定位完成后，按R1键同时按住按钮C移动显微镜对吸管</li><li>通过随时随地聚焦显微镜在其尖端的视频显示，然后按B键同时按住按钮C.一个正方形网格重新校准每个吸管的位置应该短暂地出现，指示移液管的位置确定。如果位置不正确，定位针尖上的视频显示，然后按下按钮z的中央点的同时按住按钮C。 </li></ol></li><li> 建立细胞连接配置 <ol><li>确认的利息为当前移液器细胞是正确的标记。否则，将显微镜的利益与中央红点的单元格相匹配。马克按下按钮Ÿ同时按住按钮C.按L2键同时按住按钮C定位吸管200微米远离其指定单元格的单元格，在显微镜会自动移动到相应的位置。 </li><li>调整吸管位置，它的红点相匹配。调整吸管按R1键同时按住按钮十偏移</li><li>按L1键同时按住按钮十，慢慢地接近移液器其归因细胞激活测试脉冲。 </li><li>在观察细胞的膜表面上的凹坑的形成施加的负压的简要脉冲通过按下按钮Ÿ同时按住按钮Z到允许所施加的压力，以达到细胞。约-65mV的钳制电位应该在这一点上按下按钮，L2按住按钮成立十</li></ol></li><li> 全细胞构<ol><li>一旦一个千兆密封形成为每个小区，开始通过施加负压力破裂的膜。 </li></ol></li><li> 进行录音 <ol><li>使用刺激/采集系统来执行你的录音。应用脉冲或在一个单独的细胞的脉冲串的时间和观察剩余的细胞反应来映射记录细胞间的连接。 </li></ol></li><li> 退去移液器 <ol><li>一旦录音完成后，退去吸管慢慢地从组织通过右键单击该表单选按钮。选择"退去 - &gt;移液器500μm的&#39;有退去的移液管沿其轴线不远。观察漂移的细胞电位（通过应用一些正压清除提示可帮助）。 </li><li>退去移液器所有回来的路上，设置定位速度，以&#39;快&#39;，并重复相同的操作，但选择了"全部"选项。卸下用过的移液器轻轻滑出的探头和旋从人的移液管。为了避免扭曲持有人在各自的插槽中，因为这可能会极大地与枪头位置妨碍它是有用的。 </li></ol></li></ol>

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作者宣称，他们有没有竞争的财务权益。

一个直接的问题，通常发生关于我们描述的过程的成功率。对于高成功率的准备是必不可少的。移液器必须有尖端的孔，它们足以记录细胞生物。过滤该溶液内，以避免堵塞移液器也是重要的。非常干净的，新鲜的拉移液管是另一种要求。甲二项式分布，可用于理解这些问题如何影响最终产率的最简单的模型。它是合理的期望实验者有经验和适当的设备，以达到80％或以上的记录与视觉反馈的单?...

与微米精确记录和刺激多个站点的能力是通过实验获得更好地了解神经系统非常有用。许多技术已被开发，为此，但没有允许通过膜片钳技术，对于研究的亚阈值的活性和个体的突触后电位所必需的submillivolt分辨率实现的。这里我们介绍一个12 - 电极计算机辅助膜片钳系统旨在同时记录和刺激大量单个细胞以足够的精度对神经元连接的研究的发展。而许多其它应用中，可以设想这样一个系统，它本身特别好到的突触连接的研究考虑到在一组神经元的可能的连接的数量成比例地增大到所讨论的神经元的数量的平方。因此，在具有三个电极的系统允许测试发生多达六个连接和最经常记录单1，记录12的神经元可以检测多达132连接的发生，经常观察超过一打（ 图1）。几十个连接的观测同时使得能够分析小型网络的组织和推断出网络结构的统计特性不能被探测否则为1。此外，许多细胞的精确刺激也允许招募突触后细胞2的定量。

<table><tbody><tr><td>Microscope</td><td>Olympus</td><td>BX51WI</td><td>40X Immersion Objective</td></tr><tr><td>Manipulators</td><td>Luigs Neumann</td><td>SM-5</td><td>Serial protocol used</td></tr><tr><td>Amplifiers</td><td>Axon Instruments</td><td>MultiClamp 700B</td><td>SDK used</td></tr><tr><td>Camera</td><td>Till Photonics</td><td>VS 55</td><td>BNC analog output</td></tr><tr><td>Framegrabber</td><td>Data Translation</td><td>DT3120</td><td>SDK used</td></tr><tr><td>Oscilloscopes</td><td>Tektronix</td><td>TDS 2014</td><td>Serial communication</td></tr><tr><td>Data acquisition</td><td>InstruTECH</td><td>ITC 1600</td><td></td></tr><tr><td>Data acquisition</td><td>National Instruments</td><td>PCI-6221</td><td>Library used (.dll)</td></tr><tr><td>Pressure valve</td><td>SMC</td><td>SMC070C-6BG-32</td><td></td></tr><tr><td>Pressure sensor</td><td>Honeywell</td><td>24PCDFA6G</td><td></td></tr><tr><td>Membrane pump</td><td>Schego</td><td>Optimal</td><td></td></tr></tbody></table>

a computer-assisted multi-electrode patch-clamp system

膜片钳技术是目前最行之有效的方法，用于记录电活动从单个神经元或他们的亚细胞区室。然而，实现稳定的记录，即使从单个细胞，仍具有相当复杂耗时的过程。在与高效率的信息显示结合多步骤的自动化可以极大地帮助实验者与更高的可靠性进行录音的数量较多，并在更短的时间。为了实现大规模的录音，我们得出的结论是最有效的方法是不完全使自动化过程，但为了简化实验步骤和减少人为错误的可能性，同时有效地掺入实验者的经验和视觉反馈。考虑到这些目标，我们开发了其中集中了所有必要的控制的多电极膜片钳实验中的单个接口，COMMERC计算机辅助系统ially可用的无线游戏手柄，在电脑屏幕上显示实验相关的信息和指导线索。在这里，我们描述系统使我们能够减少用于实现记录配置的时间，大幅增加成功记录大量的神经元同步的机会的不同组成部分。

按照以上描述的方法，我们成功地同时进行多达12神经元的全细胞记录，几乎增加一倍的最大数目的神经元同时膜片钳制迄今。记录大鼠的躯体感觉皮层V层锥体神经元之间的直接的突触连接的网络的例子示于图6。 的连接概率访问，作为对于给定的细胞类型间体的距离的函数的确定为感兴趣的典型的测量，可以有效地与多电极膜片钳系统1来进行。最近的?...

Watch this Scientific Journal Video about A Computer-assisted Multi-electrode Patch-clamp System at JoVE.com

A Computer-assisted Multi-electrode Patch-clamp System

多电极膜片钳记录构成一个复杂的任务。这里，我们显示了如何通过许多的实验步骤的自动化，因此能够加速导致质的提高的性能和记录的数目的过程。

计算机辅助多电极膜片钳系统

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

a-computer-assisted-multi-electrode-patch-clamp-system

Research

JoVE Journal

Neuroscience

13.7K 次观看.  Ecole Polytechnique Federale de Lausanne. 多电极膜片钳记录构成一个复杂的任务。这里，我们显示了如何通过许多的实验步骤的自动化，因此能够加速导致质的提高的性能和记录的数目的过程。 

视频: A Computer-assisted Multi-electrode Patch-clamp System

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

人类癫痫术后皮层组织的多电极阵列录音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

科研

医学

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

生物学

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

自动图像引导贴片夹在脑切片神经元研究中的应用

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

神经科学

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Patch Clamp Electrophysiology

Neuron cell membranes are populated with ion channels that control the movement of charge into and out of the cell, thereby regulating neuron firing. One extremely useful technique for investigating the biophysical properties of these channels is called patch clamp recording. In this method, neuroscientists place a polished glass micropipette against a cell and apply suction to form a high resistance seal. This process isolates a small &#8220;patch&#8221; of membrane that contains one or more ion channels. Using an electrode within the micropipette, researchers can &#8220;clamp&#8221; or control the electrical properties of the membrane, which is important for analysis of channel activity. The electrode also allows for changes in the voltage across the membrane, or the flow of ions through the membrane, to be recorded. This video begins with an overview of the principles behind patch clamp electrophysiology, an introduction to the necessary equipment, and descriptions of the various patch configurations, including whole cell, cell-attached, perforated, inside-out, and outside-out patches. Next, the key steps of a typical whole-cell patch clamp experiment are outlined, in which a current-voltage (IV) curve is generated. Finally, applications of patch clamp recording are provided to demonstrate how the biophysical properties of ion channels, cell excitability, and neuroactive compounds are evaluated in neurophysiology labs today.

Patch Clamp Electrophysiology: Principle &amp; Applications

膜片钳电生理技术

Neuron cell membranes are populated with ion channels that control the movement of charge into and out of the cell, thereby regulating neuron firing. One ...

教育

JoVE Science Education

高级生物学

神经科学的基本知识

Establishing a Whole-Cell Configuration through the Patch Clamp Method

Source: Segev, A. et al., <a href="https://app.jove.com/v/54024">Whole-cell Patch-clamp Recordings in Brain Slices.</a>&#160;J. Vis. Exp.&#160;(2016)
This video demonstrates the whole-cell patch clamp technique in a mouse brain slice. This technique establishes a stable connection between micropipette and neuron membrane, which is further disrupted to establish a whole-cell configuration to study the neuron&#39;s health and physiological responses.

通过膜片钳方法建立全细胞配置

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Slice ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Amphotericin-B Mediated Perforated Patch-Clamp Analysis: An Electrophysiological Technique to Study Ionic Currents in Urinary Bladder Cells

Source: Malysz, J. et al. <a href="https://www.jove.com/video/59884" target="_blank">Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents.</a> J. Vis. Exp. (2020)
This video demonstrates the characterization of cationic currents from transient receptor potential melastatin type 4 (TRPM4) ion channels in detrusor smooth muscle cells using amphotericin-B mediated perforated patch-clamp technique. The technique helps evaluate the biophysical and pharmacological properties of ion channels.

两性霉素 B 介导的穿孔膜片钳分析:一种研究膀胱细胞中离子电流的电生理技术

Source: Malysz, J. et al. Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of ...

癌症研究

Urinary Tract Cancer

In vitro studies

Whole-Cell Patch Clamp Electrophysiology: A Method to Study Electrical Properties of Neurons

Source: Roy, B., et al. <a href="https://www.jove.com/v/50551/" target="_blank">Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells</a>. J. Vis. Exp. (2013).
This protocol describes a technique called whole-cell patch clamp electrophysiology, which is a method to study electrical properties of Mauthner neurons and other reticulospinal cells in zebrafish embryos.

全细胞膜片钳电生理学:一种研究神经元电特性的方法

Source: Roy, B., et al. Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells. J. Vis. Exp. (2013).
This protocol describes a technique called ...