Name: application of automated image-guided patch clamp for the study of neurons in brain slices
Uploaded: 2017-07-31T14:00:00.000+00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices at JoVE.com

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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	<li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annu Rev Physiol. 46, 455-472 (1984).</li><li>Collins, M. D., Gordon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+liposome+preparation+for+imaging+and+patch-clamp+electrophysiology.">Giant liposome preparation for imaging and patch-clamp electrophysiology.</a> J Vis Exp. (76), (2013).</li><li>Kodandaramaiah, S. B., Franzesi, G. T., Chow, B. Y., Boyden, E. S., Forest, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+whole-cell+patch-clamp+electrophysiology+of+neurons+in+vivo.">Automated whole-cell patch-clamp electrophysiology of neurons in vivo.</a> Nat Methods. 9 (6), 585-587 (2012).</li><li>Desai, N. S., Siegel, J. J., Taylor, W., Chitwood, R. A., Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MATLAB-based+automated+patch-clamp+system+for+awake+behaving+mice.">MATLAB-based automated patch-clamp system for awake behaving mice.</a> J Neurophysiol. 114 (2), 1331-1345 (2015).</li><li>Kodandaramaiah, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+operation+of+the+autopatcher+for+automated+intracellular+neural+recording+in+vivo.">Assembly and operation of the autopatcher for automated intracellular neural recording in vivo.</a> Nat Protocols. 11 (4), 634-654 (2016).</li><li>Wu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+autopatching+with+automated+pipette+and+cell+detection+in+vitro.">Integration of autopatching with automated pipette and cell detection in vitro.</a> J Neurophysiol. 116 (4), 1564-1578 (2016).</li><li>Perin, R., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computer-assisted+multi-electrode+patch-clamp+system.">A computer-assisted multi-electrode patch-clamp system.</a> J Vis Exp. (80), e50630 (2013).</li><li>Fertig, N., Blick, R. H., Behrends, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+cell+patch+clamp+recording+performed+on+a+planar+glass+chip.">Whole cell patch clamp recording performed on a planar glass chip.</a> Biophys J. 82 (6), 3056-3062 (2002).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+patch-pipettes+and+sharp+electrodes+with+a+programmable+puller.">Making patch-pipettes and sharp electrodes with a programmable puller.</a> J Vis Exp. (20), (2008).</li><li>Segev, A., Garcia-Oscos, F., Kourrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+Patch-clamp+Recordings+in+Brain+Slices.">Whole-cell Patch-clamp Recordings in Brain Slices.</a> J Vis Exp. (112), (2016).</li><li>Campagnola, L., Kratz, M. B., Manis, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACQ4:+an+open-source+software+platform+for+data+acquisition+and+analysis+in+neurophysiology+research.">ACQ4: an open-source software platform for data acquisition and analysis in neurophysiology research.</a> Front Neuroinform. 8 (3), (2014).</li><li>Kolb, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaning+patch-clamp+pipettes+for+immediate+reuse.">Cleaning patch-clamp pipettes for immediate reuse.</a> Sci Rep. 6, (2016).</li></ol>

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。 与现有的体外膜片钳方法相比，该系统在几个方面取得了显着的进步。它最大限度地减少人为干预该系统自动化了膜片钳实验中的大部分步骤，使需求最小化为人为干预而设。剩下的一些手动步骤，包括低/高倍率显微镜镜片之间的切换，可以使用附加的电动硬件自动进行。 膜片钳方法提高了吞吐量。使用该系统的贴片钳实验在每个试验中获得更高的成功率和更短的时间，有助于总体生产量的显着增加。用于荧光细胞检测和移液管尖端检测的计算机视觉算法非常强大，误差率非常低。移液管尖端检测的平均误差为1.6μm，荧光细胞检测的假阳性率为4.9％±2.25％。传统的手工修补和自动修补之间的详细比较已取得6。 实验的详细文件是可能的。每个试验的补丁日志可以在事后保存和分析。这样详细文档以前不可用于手动修补。这允许在独特的实验条件，细胞类型，物种和切片制剂中的修补实验的系统分析。 该方法显示与标准体外膜片钳设备的兼容性。如本手稿所示，我们的系统旨在增强现有的体外膜片钳装置，使他们有能力进行自动修补。与平面贴片方法不同，该系统适用于已经进行手动贴片钳的实验室，以最小的成本转换其设备。同时，仍然可以使用相同的系统手动或半自动修补。 由于上述系统的适应性，当系统首次设置时，实验者需要连接硬件和配置软件。可能会导致问题从错误的端口分配和驱动程序库不足以控制某些硬件。故障排除时请参考步骤1.2 - 1.4。 与现有系统的部分自动化相比，该系统实现了常规体外补片夹紧急性脑切片（和其他体外准备）的最大自动化水平。这对于所有的步骤如此，从小区检测到移液器校准到修补7,11。唯一的瓶颈是在路径之间填充和更换补片移液器的手动过程。在补丁吸管的再利用最近的事态发展有可能解决这个问题12。除了切片准备的质量之外，不成功试验的最常见原因源于机械手的机械误差以及切片在腔室中的运动。这些限制是我们无法控制的当前系统正在努力实现闭环，实时检测和控制移液器移动以解决这个问题。 对于未来的发展，我们有兴趣将目前的荧光细胞检测能力扩展到DIC光学下的通用细胞检测。

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><tbody><tr><td>CCD Camera</td><td>QImaging</td><td>Rolera Bolt</td><td></td></tr><tr><td>Electrophysiology rig</td><td>Scientifica</td><td>SliceScope Pro 2000</td><td>Include microscope and manipulators. The manufacturer provided manipulator control software demonstrated in this manuscript is &amp;ldquo;Linlab2&amp;rdquo;.</td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>MultiClamp 700B</td><td>computer-controlled microelectrode amplifier</td></tr><tr><td>Digitizer</td><td>Molecular Devices</td><td>Axon Digidata 1550</td><td></td></tr><tr><td>LED light source</td><td>Cool LED</td><td>pE-100</td><td>488 nm wavelength</td></tr><tr><td>Data acquisition board</td><td>Measurement Computing</td><td>USB1208-FS</td><td>Secondary DAQ.&lt;br/&gt; See manual at : http://www.mccdaq.com/pdfs/manuals/USB-1208FS.pdf</td></tr><tr><td>Solenoid valves</td><td>The Lee Co.</td><td>LHDA0531115H</td><td></td></tr><tr><td>Air pump</td><td>Virtual industry</td><td>VMP1625MX-12-90-CH</td><td></td></tr><tr><td>Air pressure sensor</td><td>Freescale semiconductor</td><td>MPXV7025G</td><td></td></tr><tr><td>Slice hold-down</td><td>Warner instruments</td><td>64-1415 (SHD-40/2)</td><td>Slice Anchor Kit, Flat for RC-40 Chamber, 2.0 mm, 19.7 mm</td></tr><tr><td>Python</td><td>Anaconda</td><td>version 2.7 (32-bit for windows)</td><td>https://www.continuum.io/downloads</td></tr><tr><td>Screw Terminals</td><td>Sparkfun</td><td>PRT - 08084</td><td>Screw Terminals 3.5 mm Pitch (2-Pin)</td></tr><tr><td>(2-Pin)</td><td></td><td></td><td></td></tr><tr><td>N-Channel MOSFET 60 V 30 A</td><td>Sparkfun</td><td>COM - 10213</td><td></td></tr><tr><td>DIP Sockets Solder Tail - 8-Pin</td><td>Sparkfun</td><td>PRT-07937</td><td></td></tr><tr><td>LED - Basic Red 5 mm</td><td>Sparkfun</td><td>COM-09590</td><td></td></tr><tr><td>LED - Basic Green 5mm</td><td>Sparkfun</td><td>COM-09592</td><td></td></tr><tr><td>DC Barrel Power Jack/Connector (SMD)</td><td>Sparkfun</td><td>PRT-12748</td><td></td></tr><tr><td>Wall Adapter Power Supply - 12 V DC 600 mA</td><td>Sparkfun</td><td>TOL-09442</td><td></td></tr><tr><td>Hook-Up Wire - Assortment (Solid Core, 22 AWG)</td><td>Sparkfun</td><td>PRT-11367</td><td></td></tr><tr><td>Locking Male x Female x Female Stopcock</td><td>ARK-PLAS</td><td>RCX10-GP0</td><td></td></tr><tr><td>Fisherbrand Tygon S3 E-3603 Flexible Tubings</td><td>Fisher scientific</td><td>14-171-129</td><td>Outer Diameter: 1/8 in.&lt;br/&gt; Inner Diameter: 1/16 in.</td></tr><tr><td>BNC male to BNC male coaxial cable</td><td>Belkin Components</td><td>F3K101-06-E</td><td></td></tr><tr><td>560 Ohm Resistor (5% tolerance)</td><td>Radioshack</td><td>2711116</td><td></td></tr><tr><td>Picospritzer</td><td>General Valve</td><td>Picospritzer II</td><td></td></tr></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 ）。 图3a是修补神经元的差分干涉对比度（DIC）图像。全细胞配置通过步骤2.5 - 2.6中的自动修补协议实现，并通过步进电流注入诱导的动作电位（ 图3c ）验证， 1"&gt;为了演示额外的"命令序列"功能，我们在修补细胞时，将500毫克KCl 200毫秒传送到脑切片上的三个位置（ 图4 ）。首先，我们在脑切片上选择了3个位置:一个关闭将该坐标存储在"Memory Positions"GUI中，坐标被加载到"Unit1"下的"Command Sequence"（命令序列）GUI中，该单元是KCl-我们设置左列中的命令发送一个+ 5-V TTL信号500 ms，接着是0 V，持续10 s（ 图4a ），从辅助DAQ板上的A口通道0，其连接到数字化仪"开始触发"输入， 图4c显示了修补的细胞是正常的尖峰神经元，药物应用移液器（单元1）穿过三个选定的位置a自动（ 图4b ），我们在电压钳下记录每个应用10秒（ 图4d ）。 图4d中的迹线的颜色对应于图4b中的边框颜色。当KCl在细胞中膨化时，观察到大的向内电流，随着KCl扩散而缓慢减小。将红色荧光染料加入到KCl溶液中以指示药物递送的空间分布，并使用组合的DIC和荧光成像成像。本实验说明了我们的系统通过TTL信号控制机械手/显微镜运动和外部硬件的简便性和灵活性。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/56010/56010fig1.jpg" /> 图1.压力控制单元A:印刷电路板（PCB）用于连接阀门，压力传感器和气泵。左侧显示了PCB上的详细信息，在协议中提及的输出的标记位置。右侧显示了PCB与气泵，USB端口和管路之间的连接。 B: PCB的电路图。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56010/56010fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/56010/56010fig2.jpg" /> 图2.自动绘图GUI。协议中提到的按钮以红色方块显示并编号。 1:开始校准，2:保存校准，3:负载校准，4:辅助校准，5:检测单元，6:补丁控制，7:转到（目标单元坐标）和8:补丁。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56010/56010fig2large.jpg" target="_blank">请点击此处查看此图的较大版本</a>。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/56010/56010fig3.jpg" /> 图3.补丁ChR2-YFP阳性细胞的实例。 A: DIC光学下40倍放大。 B:面板A中相同电池的荧光图像（488nm的LED照明）。 C:在一系列超极化和去极化步骤电流注入期间来自修补电池的电流钳记录。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56010/56010fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/56010/56010fig4.jpg" /> 图4.进行自动药物递送实验。 A:加载到"命令序列"GUI的所选位置。左侧列显示坐标列表和右侧列显示每个位置的TTL信号形式的命令列表。 B:在药物应用实验期间对应于三个选定位置的截图。第1单元是含KCl的移液管，第2单元是补丁移液器。将KCl溶液与红色荧光染料混合，以便可视化。通过组合DIC和荧光成像获得图像。 C:步进电流注射显示正常的尖峰神经元。 D:在三个位置局部应用500mM KCl溶液的电压钳记录痕迹。当KCl施用接近修补细胞时，从试验中记录具有向内电流的红色痕迹。红色箭头表示KCl应用的时间。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56010/56010fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1"fo:keep-together.within-page ="1"fo:keep-with-next.within-page ="always"&gt; <tbody><tr><td> PCB上的插座 </td><td> DAQ板上的端口名称 </td><td> 端口＃在DAQ板上 </td><td> 备注 </td></tr><tr><td> DOUT V1 </td><td>端口A通道1 </td><td> 22 </td><td>控制阀1 </td></tr><tr><td> DOUT V2 </td><td>端口A通道2 </td><td> 23 </td><td>控制阀2 </td></tr><tr><td> DOUT P </td><td>端口A通道3 </td><td> 24 </td><td>控制气泵</td></tr><tr><td> GR </td><td>地面</td><td> 29 </td><td>地面</td></tr></tbody></table> 表1.印刷电路板（PCB）到二次数据采集（DAQ）板连接配置。使用此表将PCB输出（左起第一列）连接到DAQ板上的端口（左侧第二列））。辅助DAQ上的端口名称和编号是指单端模式。 <img alt="表2" src="/files/ftp_upload/56010/56010table2.jpg" /> 表2.从压力控制单元到移液器支架的管道连接。对于每个连接，使用软管连接相应的端口，用灰色框突出显示（参见材料表 ）。

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.5K 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

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 ...

科研

神经科学

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 ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

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 ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

分层膜片钳技术在学习诱导可塑性分析中的应用

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

全细胞膜片电生理方法对突触倍数的评价

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

Establishing a Whole-Cell Voltage-Clamp Configuration for Electrophysiological Recordings in Brain Slices

Source: Sunstrum, J. K., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59461">Evaluation of synaptic multiplicity using whole-cell patch-clamp electrophysiology.</a> J. Vis. Exp. (2019)
This video demonstrates positioning a fine-pointed pipette near brain slices, forming a seal with a neuronal cell. Suction breaks the cell membrane, thereby creating a whole-cell patch clamp that enables current measurements for electrophysiology experiments.

为脑切片中的电生理记录建立全细胞电压钳配置

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

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Automated Image-Guided Patch Clamp Technique for Studying Neurons in Brain Slices

Source: Wu, Q., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56010">Application of automated image-guided patch clamp for the study of neurons in brain slices.</a> J. Vis. Exp. (2017).
In this video, brain slices are immobilized in a recording chamber, and cells of interest are located and focused on using a computer-controlled microscope. The automated image processing system then precisely positions a patch pipette to seal the cell membrane and monitor electrical signals.

用于研究脑切片中神经元的自动图像引导膜片钳技术

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. System Setup

	Construct ...

Paired Patch Clamp Recording of the Neuronal Connections in Acute Brain Slices

Source: Qi, G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52358">Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings.</a> J. Vis. Exp. (2015)
This video demonstrates the procedure to map synaptic connections by securing a brain slice, positioning pipettes to achieve whole-cell configurations, and stimulating presynaptic neurons to record postsynaptic responses, thereby elucidating neuronal connectivity.

急性脑切片中神经元连接的配对膜片钳记录

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Paired Patch-Clamp ...

Simultaneous Optical and Electrophysiological Monitoring of Neuronal Cells in Brain Slices

To place the multi-channel probe in the ex vivo brain tissue slide, perfuse bubbled experimental ACSF at three to six milliliters per minute, and transfer the brain slice containing the area of interest onto the mesh grid in the microscope perfusion chamber. Anchor the brain slice with a platinum harp. Rotate the mesh grid so that the line of electrode contacts on the distal end of the multichannel probe is approximately perpendicular to the PL surface.
Under broad-field illumination and fine control of the micromanipulator, lower the multichannel probe toward the surface of the slice. Engage the appropriate filter cube for visualization of the fluorescent reporter protein expressed in axon terminals of cortical afferents.
If necessary, rotate the slice to more precisely align the probe with the PL surface. Position the probe just above the plane of the slice, 200 micrometers short of the final target position along the x-axis, leaving at least one channel outside of the area of tissue being recorded. Slowly, insert the probe into the slice along its longitudinal axis.
To minimize damage to the tissue, only advance the probe to the extent that the sharp tips are just visible below the tissue surface. Switch experimental ACSF source to bagged control solution, and identify the fluorescently labeled cell for targeted patch-clamp recording. Restrict the aperture to the smallest diameter, and engage the high-power water-immersion objective, taking care to avoid contact between the multichannel probe and objective lens. Bring the tissue into focus.
Center the light over an area of tissue adjacent to, but not overlapping the multi-channel probe. Engage the appropriate filter set to allow imaging of cells expressing the Cre-dependent fluorescent marker. Raise the objective lens to create ample space for lowering a patch pipette.
Load a patch pipette with internal solution and mounted on the electrode holder. Use a one-milliliter syringe to apply positive pressure corresponding to approximately 0.1 milliliter air. Lower the patch pipette into the solution, bringing the tip into focus under visual guidance, and obtain whole-cell recording from the targeted cell.