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 border="1">
	<tbody>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>40X Immersion Objective</td>
		</tr>
		<tr>
			<td>Manipulators</td>
			<td>Luigs &#38; 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

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 ...

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 ...

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 ...

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 ...

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.