我们非常感谢Whitehall基金会的财政支持。我们要感谢萨缪尔·基辛格的宝贵意见。

系统设置<ol><li>构造压力控制单元。 <ol><li>根据电路图组装压力控制单元（ 图1 ）。将必要的部件焊接到根据电路原理图制造的印刷电路板（PCB）上（ 图1b ）。使用标准电阻器，LED，金属氧化物半导体场效应晶体管（MOSFET），电容器和连接器（见材料表 ）。焊接电磁阀到PCB上。使用电线将气泵和空气压力传感器连接到PCB。 注意:建立压力控制单元需要大约2小时，所有必要的部件都可用。 </li></ol></li><li>连接二次数据采集（DAQ）板。 <ol><li>将印刷电路板的数据输出连接到DAQ板，如表1所示 。 注意:DAQ板将会通过我以"单端模式"运行。端口图可以在用户手册中找到（参见材料表 ）。 </li><li>将"AIn Pr S"连接到模拟输入（AI）通道之一，将"R-Gr"连接到辅助DAQ板上的模拟接地之一。 </li><li>将放大器的主输出端连接到辅助DAQ板的模拟地之间的AI通道和地之一。 注意:标准BNC电缆可用于连接放大器的主输出。 </li><li>剥去另一端并将正信号（ 即铜芯）连接到AI通道和地（ 即芯周围的细线）到模拟地。如果使用多个补丁通道，则对第二个通道重复此步骤。 注意:DAQ板的模拟输入将在后面的步骤中进行配置。 </li><li>将电源连接到辅助DAQ板的电源输出。使用单独的12 V电源用于泵。 </li></ol></li><li>连接油管。 <ol><li>根据表2连接气泵和两个阀。在最后一步中，使用3路连接器连接来自阀门2顶端口，压力传感器和移液器支架的软管。 </li><li>如果使用两个移液器，请向连接到移液器支架的管道上添加另一个3通连接器。在修补时，在使用中的阀门和移液器之间手动切换。 </li></ol></li><li>安装Autopatcher IG。 注意:系统要求:Autopatcher IG仅在运行Windows 7的PC上进行测试。尚未验证其他操作系统。所描述的过程特别适用于材料表中列出的硬件。 <ol><li>从GitHub下载Autopatcher-IG（https://github.com/chubykin/AutoPatcher_IG）。 </li><li>安装Python（有关版本和下载地址的资料表 ）。 </li><li>卸载PyQt4在命令行终端中键入"pip uninstall PyQt4"的库。 注意:系统使用旧版本的PyQt4库来实现与Qwt和Opencv库的兼容性。 </li><li>从历史轮文件（http://www.lfd.uci.edu/~gohlke/pythonlibs/）安装Python库。查找以下文件:Numpy（pymc-2.3.6-cp27-cp27m-win32.whl），Opencv（opencv_python-2.4.13.2-cp27-cp27m-win32.whl），Pyqt（PyQt4-4.11.4-cp27-none -win32.whl）和Qwt（PyQwt-5.2.1-cp27-none-win32.whl）。 <ol><li>要安装车轮文件，请转到保存文件的目录，然后键入"pip install *** wheelfilename ***。whl"。用文件的实际名称替换"*** wheelfilename ***"。 注意:车轮文件名中的"cp27"表示Python 2.7，"win32"表示Windows 32位。如果"win32"不起作用，请尝试"win64"。 </li></ol></li><li>要控制CCD摄像头，请下载并安装安装程序用于64位（https://www.qimaging.com/support/software/）。然后下载MicroManager for 64-bit（https://micro-manager.org/wiki/Download_Micro-Manager_Latest_Release）来控制相机的Python。 </li><li>要控制操纵器和显微镜平台，请安装制造商提供的控制软件。 注意:通过这样做，也安装了控制操纵器所必需的驱动程序。安装包通常在CD-ROM中提供。 </li><li>要控制辅助DAQ板，请随购买DAQ板一起安装光盘中的通用库。 </li></ol></li><li>配置Autopatcher IG的硬件。 <ol><li>通过USB端口将显微镜台和机械手控制器连接到计算机。 </li><li>将COM端口号分配给单元0:显微镜台，单元1:左操纵器和单元2:右操纵器，按此顺序，在"配置"文件夹中的"ports.csv"配置文件中。大号保存ports.csv文件中的其他参数（ 即 "SCI"和"1"）不变。 注意:通过运行制造商提供的机械手配置软件可以找到COM端口号信息。转到"设置"选项卡，选择"设置"和"运动"页面，并阅读顶部每个选项卡的标签。或者，该信息可以在PC设备管理器中找到。 </li><li>在DAQ板上分配压力传感器和补丁通道1和2（对应于单元1和2）的模拟输入通道号。在"配置"文件夹中的"DAQchannels.csv"文件中输入通道号。 注意:建议使用记事本应用程序而不是电子表格打开.csv文件，因为它可能会在保存更改时更改信息。 </li></ol></li><li>运行Autopatcher IG。 <ol><li>打开放大器，显微镜控制器和机械手控制器。 Ensu放大器软件正在运行。 </li><li>从命令行终端用Python运行Autopatcher IG，如下所示:首先，在安装了Autopatcher IG的目录下，更改目录（命令"cd"），在命令行终端键入"python Autopatcher_IG.pyw"，然后点击"回车键。 注意:在运行Autopatcher IG之前，不要运行机械手控制软件，因为它将占用显微镜平台和操纵器，导致Autopatcher IG无法找到硬件。如果需要控制其他模块（ 如在线加热器） ， Autopatcher IG完全启动后，可以运行机械手控制软件。 </li></ol></li><li>校准主移液器。 <ol><li>如前所述拉拉式移液器9 。用内部溶液填充拉拔的玻璃移液器，并将其装载到头架上。 注意:空玻璃移液器具有不同的对比度显微镜可能导致校准不准确。 </li><li>将移液器吸头移至显微镜视野并使其聚焦。如果拨号盘用于移动操纵器和/或显微镜台，请按键盘上的"z"来更新坐标。 注意:如果键盘（显微镜平台:A / D - x轴，W / S-y轴，R / F-z轴;操纵器:H / K - x轴，U / J-y轴，O / L-z轴，1/2单位数）用于控制运动，因为坐标将被实时更新。 </li><li>单击主图形用户界面（GUI）上的"开始校准"按钮，以获取装有移液器的相应单元（ 图2 ）。 注意:校准完成后将出现一个弹出窗口。 注意:校准将自动进行，大约需要3.5分钟。点击相同的按钮（现在切换为"取消校准"af启动校准）将中止校准尝试。 </li><li>通过点击主界面底部的"保存校准"来保存校准（可以保存两个操纵器的当前校准，并可以在将来加载）。 注意:低（4或10X）和高（40X）放大倍率下的视野必须对齐，以便二次校准才能正常工作。请参阅使用的光学系统的用户手册进行校准步骤。 </li></ol></li></ol> 2.自动补丁程序<ol><li>准备急性脑切片，如前所述10 。 </li><li>准备贴片钳的玻璃移液器。 </li><li>将一个脑切片置于记录室的中心。用切片按压或"竖琴"来稳定切片。 </li><li>检测荧光细胞。 <ol><li>找到4X镜头下的感兴趣区域。通过打开cli移动显微镜平台ck-to-move（"CTM"）模式，并点击感兴趣区域的中心。或者，使用键盘移动显微镜平台（A / D - x轴，W / S - Y轴，R / F-z轴）。 </li><li>切换到高倍率镜头并通过在Z轴上移动显微镜来调整对焦，使用键盘上的R / F。 注意:建议调整水浴水平，使低低和高倍率镜头下的焦平面在z轴上相同或相似。 </li><li>单击主GUI界面"单位0."上的"检测单元格"按钮。如果设置的LED或激光光源不能被TTL信号控制，请手动打开LED /激光器;当单元格检测完成时，将出现一个弹出窗口。 <ol><li>如果需要，关闭LED /激光;单元坐标列表将出现在"内存位置"GUI中。通过点击坐标旁边的"X"按钮从列表中删除不需要的单元格。 </li><li>或者，如果目标单元未被荧光标记，请单击主GUI上的"鼠标模式"。点击感兴趣的单元格;具有数字的黄点将出现在单元格上，单元格的坐标将出现在"存储位置"GUI中。 </li></ol></li></ol></li><li>校准二次移液器。 <ol><li>用内部溶液填充1/3的玻璃移液管。将移液器装入附着在头架上的移液器支架上。 </li><li>使用低倍率镜头。使用移液器进入视野并使用小键盘（H / K - x轴，U / J-y轴，O / L-z轴）调整对焦。使用"1"和"2"在单元1和单元2之间切换。 </li><li>单击"加载校准"加载主校准。在正在使用的设备下的主GUI上单击"辅助校准"按钮。例如，如果单元2在使用中，请单击"单元2"中的"辅助校准"按钮lumn。按照弹出窗口的说明切换到高倍率镜头。 </li></ol></li><li>修补目标细胞。 <ol><li>确保"MultiClamp"（ 即放大器）软件正在运行。点击"补丁控制"按钮打开"补丁控制"GUI;打开此GUI可能需要几分钟的时间。 </li><li>通过在主GUI"单元0"列上选中"40X"，使用40倍放大镜。点击"记忆位置"GUI坐标列表上感兴趣的单元格旁边的"转到"按钮;显微镜将移动到细胞。 </li><li>单击主GUI中正在使用的设备的CTM按钮，以便在鼠标点击后启用移动。点击感兴趣的单元格;移液器尖端将移动到细胞。 </li><li>单击"补丁控制"GUI上的"补丁"按钮。 注意:自动修补开始，可以监控压力和电阻"补丁控制"GUI。 <ol><li>使用"单位1选择"按钮切换两个单元之间的输入信号。 注意:系统将接近目标细胞，施加负压，匹配细胞膜电位，并根据一系列压力和电阻阈值和逻辑检测gigaseal形成。 </li><li>通过单击"补丁控制"GUI上的各个按钮，可以随时处理自动进程。例如，再次单击"补丁"按钮以取消补丁试用，并单击"下一个阶段"将修补过程推进到下一步，而不管阈值如何。 注意:弹出窗口会在用户形成gigaseal时通知用户，并提供应用zap以及大负压的选项。 </li></ol></li><li>选择"是"以合并Zap和吸力进入。或者，选择"否"仅吸入。 <br/&gt;注意:当一个成功的全单元格修补程序完成后，弹出窗口将提示用户保存实验修补程序日志。 </li><li>保存实验补丁日志。 注意:如果修补试用不成功，弹出窗口将通知用户，修补程序将重置。 </li><li>回到步骤2.4，并用不同的单元格重复这些步骤。 </li></ol></li><li>优化修补阈值（可选）。 注意:初始移液器电阻范围的阈值，正负压，gigaseal阻力等 。可以从配置文件修改。 <ol><li>在系统安装目的地的"配置"文件夹中打开"PatchControlConfiguration.csv"文件。更改对应于每个阈值的数字。不要更改值的名称;这将导致系统中无法识别的条目。 </li><li>通过按"Ct。"来立即实现新的阈值rl + L"，而不重新启动程序;重新启动程序将从文件中实现最新的阈值。 </li></ol></li></ol>执行录音注意:一旦成功完成补丁，计算机控制的微电极放大器中的模式将被自动移动软件自动设置为电流钳（"IC"）。全细胞膜片钳记录可以使用选择的记录软件进行（该系统不包括记录功能）。如果识别出多个靶细胞，则在完成记录之后，返回步骤2.4并尝试另一个细胞。 <ol><li>使用picospritzer（可选）进行自动局部药物应用实验。 注意:在这里，以本地药物应用实验为例，描述如何使用额外的"命令序列"功能通过TTL信号控制外部硬件。 <ol><li>连接港口nnel 0和辅助DAQ板上的地线使用剥离的BNC电缆（如步骤1.2.3中所述）在数字化仪上启动触发输入。将数字转换器上的一个数字输出通道连接到picospritzer上的外部触发器。 </li><li>根据用户手册准备picospritzer。将picospritzer空气输出连接到附着在显微操纵器上的药物吸头支架。 </li><li>加载一个充满选择药物的移液器。将其连接到移液器支架。校准移液器，如步骤1.7所述。 </li><li>按照步骤2.6所述修补单元后，通过鼠标点击"鼠标模式"（从主GUI切换）中的相机视图GUI，选择所需的药物输送位置。或者，使用"网格"GUI设计网格，网格中的每个像素作为目标位置之一。 注意:通过使用鼠标拖动可以在摄像机视图GUI中操作网格。 </li><li>在"命令序列"ence"GUI，选择安装药物移液管的机械手单元，点击"加载鼠标点"或"加载网格点"，导入药物输送的所有坐标。 <ol><li>单击每个坐标条目，编辑右栏中的特定命令TTL信号。在第一个命令条目中，单击最右边的数字将其从"0"变为"1"，发送+ 5-V TTL信号。将时间（T）设置为所需的TTL信号持续时间（以ms为单位）。 </li><li>在第二个命令条目中，将所有数字设置为"0"，并将T设置为试验记录长度的持续时间。编辑所有坐标条目的命令。如果需要多个TTL信号，请单击"+"添加命令条目。 注意:如果需要，8位数字表示辅助DAQ上的端口A通道0-7（端口1 - 3被泵和两个阀占用）可以切换。 </li></ol></li><li>在数据采集模块中创建一个记录协议扫描的开始是由外部启动触发器触发的。编辑协议以在期望的时间递送药物。 </li><li>在"命令序列"GUI中，单击左侧的"运行"运行所有坐标。或者，单击右侧的"运行"，仅运行所选序列。 注意:移液器将移动到每个坐标并执行定义的TTL信号以开始记录扫描。 </li></ol></li></ol>

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美国序列号:15 / 353,719的非临时专利申请"系统和方法用于自动化图像引导电泳钳式电泳学"，于2016年11月16日提交。编号:PRF 67270-02。

在这里，我们描述了体外自动图像引导膜片钳记录的方法 。这个过程的关键步骤总结如下。首先，计算机视觉用于使用通过显微镜获取的一系列图像来自动识别移液管尖端。然后将该信息用于计算显微镜和机械手坐标系之间的坐标变换函数。计算机视觉用于自动检测荧光标记的细胞并识别其坐标。这些步骤集成了移液器瞄准和使用开源Python编程语言PyQT和OpenCV库的自动修补算法。 ...

Neher和Sakmann于20世纪70年代首先研发了膜片钳技术，研究了可兴奋膜1的离子通道。从那时起，斑块夹紧已被应用于细胞，突触和电路水平的许多不同受试者的体外和体内研究 - 在许多不同的细胞类型，包括神经元，心肌细胞，非洲爪蟾卵母细胞和人造脂质体2 。该过程涉及正确识别和靶向感兴趣的细胞，复杂的显微操纵器控制将贴片移液管移动到细胞附近，在适当的时间向移液管施加正压和负压以建立紧密的基底膜，并建立一个全单元补丁配置。贴片钳通常手动进行，需要大量培训才能掌握。即使是经验丰富的研究人员钳夹，成功率相对较低。最近，已经进行了几次尝试来实现膜片钳实验的自动化。两个主要策略已经发展到完成自动化:增加标准膜片钳设备，从头开始自动控制修补过程和新设备和技术的设计。前者的策略是适用于现有的硬件，并且可以在各种膜片钳应用中使用，包括在体内盲膜片钳3，4，5， 体外急性脑切片的膜片钳，器官切片培养物中，培养解离的神经元6 。它可以同时使用多个显微操纵器来询问复杂的局部电路7 。平面贴片方法是新开发策略的一个例子，可以实现高吞吐量同时p用于药物筛选目的的悬浮液中的细胞的atch夹钳8 。然而，平面贴片方法不适用于所有细胞类型，特别是具有长过程的神经元或包含广泛连接的完整电路。这限制了其应用来映射神经系统的复杂电路，这是传统膜片钳技术的关键优势。 我们开发了一种通过增加标准膜片钳硬件在体外自动化手动膜片钳制程的系统。我们的系统Autopatcher IG提供自动移液器校准，荧光细胞靶标识别，移液管移动自动控制，自动全细胞修补和数据记录。该系统可以自动获取不同深度的脑片的多个图像;使用计算机视觉分析他们;并提取信息，包括荧光标记细胞的坐标。那么这个信息就可以了用于目标和自动补丁感兴趣的细胞。该软件是使用Python编写的，这是一种免费的开源程序设计语言，它使用几个开源库。这确保了其他研究人员的可及性，并提高了电生理实验的重现性和严谨性。该系统具有模块化设计，使得额外的硬件可以轻松地与当前演示的系统进行接口。

<table width="747">
	<tbody>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">CCD Camera</td>
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			<td style="width:256px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="width:231px;height:101px;">Electrophysiology rig</td>
			<td style="width:115px;">Scientifica</td>
			<td style="width:147px;">SliceScope Pro 2000</td>
			<td style="width:256px;">Include microscope and manipulators. The manufacturer provided manipulator control software demonstrated in this manuscript is &#8220;Linlab2&#8221;.</td>
		</tr>
		<tr height="41">
			<td height="41" style="width:231px;height:41px;">Amplifier</td>
			<td style="width:115px;">Molecular Devices</td>
			<td style="width:147px;">MultiClamp 700B</td>
			<td style="width:256px;">computer-controlled microelectrode amplifier</td>
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			<td height="41" style="width:231px;height:41px;">Digitizer</td>
			<td style="width:115px;">Molecular Devices</td>
			<td style="width:147px;">Axon Digidata 1550</td>
			<td style="width:256px;"></td>
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		<tr height="55">
			<td height="55" style="width:231px;height:55px;">LED light source</td>
			<td style="width:115px;">Cool LED</td>
			<td style="width:147px;">pE-100</td>
			<td style="width:256px;">488nm wavelength</td>
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			<td height="98" style="width:231px;height:98px;">Data acquisition board</td>
			<td style="width:115px;">Measurement Computing</td>
			<td style="width:147px;">USB1208-FS</td>
			<td style="width:256px;">Secondary DAQ. 
			See manual at : http://www.mccdaq.com/pdfs/manuals/USB-1208FS.pdf</td>
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			<td height="21" style="width:231px;height:21px;">Solenoid valves</td>
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			<td style="width:147px;">LHDA0531115H</td>
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		<tr height="41">
			<td height="41" style="width:231px;height:41px;">Air pump</td>
			<td style="width:115px;">Virtual industry</td>
			<td style="width:147px;">VMP1625MX-12-90-CH</td>
			<td style="width:256px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="width:231px;height:41px;">Air pressure sensor</td>
			<td style="width:115px;">Freescale semiconductor</td>
			<td style="width:147px;">MPXV7025G</td>
			<td style="width:256px;"></td>
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			<td height="41" style="width:231px;height:41px;">Slice hold-down</td>
			<td style="width:115px;">Warner instruments</td>
			<td style="width:147px;">64-1415 (SHD-40/2)</td>
			<td style="width:256px;">Slice Anchor Kit, Flat for RC-40 Chamber, 2.0 mm, 19.7 mm</td>
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		<tr height="41">
			<td height="41" style="width:231px;height:41px;">Python</td>
			<td style="width:115px;">Anaconda</td>
			<td style="width:147px;">version 2.7 (32-bit for windows)</td>
			<td style="width:256px;">https://www.continuum.io/downloads</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:231px;height:20px;">Screw Terminals</td>
			<td height="41" rowspan="2" style="width:115px;height:41px;">Sparkfun</td>
			<td height="41" rowspan="2" style="width:147px;height:41px;">PRT - 08084</td>
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			<td style="width:115px;">Sparkfun</td>
			<td style="width:147px;">COM - 10213</td>
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		<tr height="75">
			<td height="75" style="width:231px;height:75px;">DIP Sockets Solder Tail - 8-Pin</td>
			<td style="width:115px;">Sparkfun</td>
			<td style="width:147px;">PRT-07937</td>
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		<tr height="21">
			<td height="21" style="width:231px;height:21px;">LED - Basic Red 5mm</td>
			<td style="width:115px;">Sparkfun</td>
			<td style="width:147px;">COM-09590</td>
			<td style="width:256px;"></td>
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		<tr height="21">
			<td height="21" style="width:231px;height:21px;">LED - Basic Green 5mm</td>
			<td style="width:115px;">Sparkfun</td>
			<td style="width:147px;">COM-09592</td>
			<td style="width:256px;"></td>
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			<td height="99" style="width:231px;height:99px;">DC Barrel Power Jack/Connector (SMD)</td>
			<td style="width:115px;">Sparkfun</td>
			<td style="width:147px;">PRT-12748</td>
			<td style="width:256px;"></td>
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		<tr height="99">
			<td height="99" style="width:231px;height:99px;">Wall Adapter Power Supply - 12VDC 600mA</td>
			<td style="width:115px;">Sparkfun</td>
			<td style="width:147px;">TOL-09442</td>
			<td style="width:256px;"></td>
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		<tr height="159">
			<td height="159" style="width:231px;height:159px;">Hook-Up Wire - Assortment (Solid Core, 22 AWG)</td>
			<td style="width:115px;">Sparkfun</td>
			<td style="width:147px;">PRT-11367</td>
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			<td style="width:115px;">ARK-PLAS</td>
			<td style="width:147px;">RCX10-GP0</td>
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			<td height="77" style="width:231px;height:77px;">Fisherbrand Tygon S3 E-3603 Flexible Tubings</td>
			<td style="width:115px;">Fisher scientific</td>
			<td style="width:147px;">14-171-129</td>
			<td style="width:256px;">Outer Diameter: 1/8 in. 
			Inner Diameter: 1/16 in.</td>
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			<td height="41" style="width:231px;height:41px;">BNC male to BNC male coaxial cable</td>
			<td style="width:115px;">Belkin Components</td>
			<td style="width:147px;">F3K101-06-E</td>
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			<td height="41" style="width:231px;height:41px;">560 Ohm Resistor (5% tolerance)</td>
			<td style="width:115px;">Radioshack</td>
			<td style="width:147px;">2711116</td>
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			<td height="21" style="width:231px;height:21px;">Picospritzer</td>
			<td style="width:115px;">General Valve</td>
			<td style="width:147px;">Picospritzer II</td>
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	</tbody>
</table>

application of automated image-guided patch clamp for the study of neurons in brain slices

全细胞膜片钳是衡量单细胞电性质的金标准方法。然而，由于其复杂性和对用户操作和控制的高度依赖，体外膜片钳仍然是具有挑战性和低吞吐量的技术。该手稿展示了一种用于急性脑切片体外全细胞膜片钳实验的图像引导自动膜片钳系统。我们的系统实施基于计算机视觉的算法来检测荧光标记的细胞，并使用显微操纵器和内部移液管压力控制将其定位成全自动修补。整个过程是高度自动化的，对人为干预的要求最低。实时的实验信息，包括电阻和内部移液器压力，以电子方式进行记录，以便将来进行分析和优化到不同的细胞类型。虽然我们的系统是在急性文胸的背景下描述的n切片记录，还可以应用于解离神经元，器官切片培养物和其他非神经元细胞类型的自动图像引导膜片钳。

<html>我们的系统已经测试了其在急性脑切片，分化为神经元的小鼠诱导的多能干细胞（iPSC）中的细胞的能力以及人造表达感兴趣的通道的HEK 293细胞的能力。 图3显示了使用Thy1-ChR2-YFP转基因小鼠（B6.Cg-Tg（Thy1-COP4 / EYFP）18Gfng / J）靶向视觉皮层中荧光标记的5层锥体神经元的实验。靶细胞是自动识别的绿色荧光阳性细胞之一（ 图3b...

Watch this Scientific Journal Video about Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices at JoVE.com

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

该协议描述了如何使用最近为标准体外电生理设备开发的系统进行自动图像引导的膜片钳实验。

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 ...

application-automated-image-guided-patch-clamp-for-study-neurons

Research

JoVE Journal

Neuroscience

11.4K 次观看.  Purdue University. 该协议描述了如何使用最近为标准体外电生理设备开发的系统进行自动图像引导的膜片钳实验。 

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

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 ...

科研

神经科学

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

局部应用药物研究烟碱型乙酰胆碱受体功能的小鼠脑片

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

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 ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

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

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

在脑片全细胞膜片钳记录

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

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

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

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

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

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

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

自动图像引导贴片夹在脑切片神经元研究中的应用

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NRCC的膜片钳芯片上细胞的培养和电

膜片钳电容测量和Ca^{2 +}单神经末梢成像在视网膜切片

局部应用药物研究烟碱型乙酰胆碱受体功能的小鼠脑片

隔离的感觉神经元中

计算机辅助多电极膜片钳系统

小脑颗粒神经元的遗传操作

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

在脑片全细胞膜片钳记录

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

粉扑诱导的全细胞电流在脑切片中的应用