Name: subcellular patch-clamp recordings from the somatodendritic domain of nigral dopamine neurons
Uploaded: 2016-11-02T12:30:00.000+00:00
Duration: 9 min 17 s
Description: Watch this Scientific Journal Video about Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons at JoVE.com

我感谢文森特Seutin博士的一贯支持，克里斯泰勒Gillissen和Laurent Massotte优秀的技术援助，博士让Defourny和桑德拉Ormenese与共聚焦显微镜，雅克注定博士第二Axopatch 200B放大器的礼物，GIGA-成像建议共享平台的共聚焦显微镜和软件了Imaris和斯蒂芬·弗里曼博士批判地阅读手稿。 FNRS（U.N002.13和T.N0015.13）和与之相配套的比利时大学基金会（PubliéAVEC乐竞赛德拉基金会区大学比利时）公布的 - 这项工作是由来自比利时FRS资助。

作者宣称，他有没有竞争经济利益。

这份报告描述了一个一步一步的协议实现双somatodendritic全细胞记录和当地树突录音。它是用于确定对突触后电位的时间过程离子通道（ 即，I H）的影响，并映射离子通道（Ⅰh）条沿黑质多巴胺神经元的somatodendritic域的分布，分别是有用的。导致电测量相结合， 事后组织化学恢复细胞形态。该程序是用来调查位于黑质多巴胺神经元，但可以概括为相邻黑质的GABA神经元，腹侧被盖区DA神经元或其它脑神经元。所有的步骤，也可以跟着审查黑质神经元的树突没有重要的修改表示其他离子通道。 事后可视化是用于与轴突轴承神经元特别相关树突，如黑质细胞25,26，海马东方明珠Oriens-alveus的interneurons 21或一些CA1神经元67。有趣的是，神经元共用这一功能似乎比一般认为67更常见。形态分析也揭示了电极和轴突的精确位置。后者的检测可以通过使用免疫组68,69轴突起始段（电压门控Na +通道或锚蛋白G）的表达的蛋白质的标记进行优化。 树突状记录和随后的神经元标记收集到的数据的可靠性不变地依赖于片质量。因此最大的努力需要施加到组织内保持细胞的活力。这是用柔和处理健康动物，高品质的工具和试剂，组织和冰冷的温度足够的氧合的整个切片的制备取得。稳定的拍摄条件依赖于健康的神经元的选择。在全细胞模式，串联电阻最初应尽可能低，并保持在整个实验常数。录音的稳定性还取决于高质量的机械手缺乏漂移和振动。检查以吸管持有人的连接和探头，控制了显微电缆松弛，避免温度或舞台动作的突然变化和检查机械手的机制:可以通过优化移液器稳定性可以减少这些干扰。对于双记录，甲基13,15,21已被包含在细胞内的解决方案，但葡糖9,14,25或者可使用。然而，主要的阴离子可以改变膜电位70,71和某些电压依赖性电流72。细胞内溶液可以补充与ATP，GTP和磷酸保存physiolo神经元的gical功能。另外，在移液管溶液中加入荧光染料（ 例如 ，Alexa的594或磺酰罗丹明101 41），以一个躯体记录期间形象化树突可以是有用的，例如以放置压力施加（在参考文献的图7。41）或电刺激吸管。对细胞附着的录音吸管解决方案包含一个高K +浓度，没有Na +的记录高我 小时 。值得注意的的 Na + / K +浓度比影响电流振幅10，电流11的反转电位和I H 73选通。可替代地，I H也可以用外奏10记录。然而，在该记录结构中，在通道的邻近的细胞内环境可以扰动。因此，在I的电压依赖性活化的差异&lt;在使用细胞贴附补丁和外困10获得比较电流子&gt; h的观察。神经元的肿胀膜片钳记录期间偶尔遇到，并且经常从不同的原因产生诸如水的溶液中的组合物中的低质量，强不平衡克分子渗透压浓度或pH的细胞内和细胞外溶液39或误差之间。电生理学记录的质量对回收的神经元的形态学的质量有直接的发病率。高电阻体移液器用于双重记录（6 - 10MΩ，如在6,11参考文献）和单体录音细胞附着的记录之后，以尽量减少细胞内环境14的稀释。因此，细胞贴附记录之后的全细胞体记录被保持短（&lt;10分钟）。无论从体细胞和树突移液器外面向外补丁是针对C的正确的关闭至关重要ELL膜和细胞形态的随后回收。除了细胞的结构，神经化学含量可以为记录神经元59,74来确定。例如，细胞内蛋白质酪氨酸羟化酶可以免疫标记为多巴胺神经元13的明确鉴定。 DA神经元主要集中在SN致密部，与目前在SN相当低的密度网状部，在那里它们被混合使用一个较大的数字神经元GABA 75。而多巴胺神经元的细胞体是经常比GABA神经元大时，这些细胞的视觉识别与IR电视显微镜是不确定的，由致密部的不透明度部分阻碍。克服这些限制，多巴胺神经元的预选择可通过在神经元（TH 65或DAT为多巴胺神经元，GAD的特定人群表达荧光标记的使用转基因小鼠来促进为GABA神经元）和落射荧光照明。替代地，荧光染料可被包含在体电极的溶液，以促进枝晶的可视化。增加了荧光细胞的分辨率由尼普科夫旋转盘共聚焦14,22,30或组合以红外DGC 6双光子显微镜7带来的。几个优点相比，DIC有关DGC。首先，作为不需要DIC棱镜，对IR-DGC图像可以与荧光图像49,52,53,76重叠。第二，DGC可以与光刺激和光遗传学77相结合。 切片准备的一个缺点是神经元的完整性的保护。多巴胺神经元中的三个空间平面78,79延伸的树突和因此树枝状隔室的截断不能完全在片80避免。片的取向的选择（冠状，水平或对矢状）是一个权衡。神经支配和实验设计的原点，必须考虑选择片的正确方向。 直接树突修补是用于功能性离子通道的分布图中的细胞的不同区室的技术。此外，这种技术提供了确定在通道20的功能特性的可变性。作为一种补充的离子通道的位置和密度可通过免疫组织化学在光学及电子显微镜的水平17,23来确定。这种方法还提供了确定未至的补丁移液器小口径结构信道密度的可能性。然而，这些信道可能是在一个不同的功能状态24或甚至不活动相比，那些使用膜片钳技术记录。这两种技术，因此需要获取的位置和属性的完整图象在一个特定的蜂窝区17的离子通道。与电压敏感染料的发展，电压成像已经被用于检查AP和神经元EPSPS的在多个位置81的传播。作为替代双膜片钳记录，这种方法甚至可以为薄树突不在修补移液器访问，但必要的信号，并平均化的精确校准实现。 而在SN和腹侧被盖区的多巴胺神经元被广泛经由在生理和病理上下文躯体录音调查，其树突的功能特性保持在这两个条件是未知。从树突修补已实施黑质多巴胺神经元由几组成功13,25,26和仍然是首选的剖析这些细微的亚细胞结构8的兴奋性的方法。树突状录音提供进一步opportu无穷大审议的效率和突触传递的可塑性和树突兴奋82,83的可塑性。

神经元主要是收到他们的树突突触的信息。兴奋性和从它们产生的部位扩散到积分位点，其中动作电位（AP）的被诱发，作为输出信号抑制性突触信号。在他们的方式，突触电位通过树突的结构和被动和主动膜特性之间的相互作用的影响。这些高度可变参数的组合扩大的神经元1,2的计算能力。然而，树突的小直径却阻碍了其电性能的研究。 （ - 3微米直径0.7）树突6,7膜片钳技术3的不断发展，光学4对切片准备5种方法细化过去几十年中已经非常薄启用录音。这些方法，并且，仍然主要用于检查树突的兴奋性在各种Ø˚F元8。直接树突录音是必不可少确定的功能特性在不同的神经元隔间离子通道20-22的分布9-19和差异。这些数据与免疫相结合，光学和电子显微镜检测23,24离子通道分布的必要补充。双somatodendritic录音已实施探索动作电位9,13-15,21,22,25-27并沿神经元的somatodendritic域突触电位13,16,18蔓延的传播，获取详细的被动电缆型号28- 30，并调查神经集成31的时间分辨率。 黑质（SN），是位于参与多种功能，如运动的控制下，奖励和习惯行为的编码中脑区域。多巴胺由于具体的减少在SN多巴胺（DA）神经元的损失与在帕金森氏病32患者中观察到的运动障碍有关。黑质电路由两个主要的细胞类型:多巴胺能和GABA能神经元。有趣的是，这些神经元具有区别于其他神经元的一些特定功能。大比例的DA神经元和一些GABA神经元的轴突从树突站点指示树突是多相（轴突轴承和轴突缺失树突）25,26,33起源。从树突开始，到体细胞，最后到轴突34:这些神经元的形态与神经元的典型组织，其中信息传送遵循Cajal间发射的动态偏振法因此对比。还已知多巴胺神经元从其树突35释放多巴胺，产生爆裂活动36和NMDA受体可塑性37。该dissec这些现象是化不从他们的起始位点直接录音难以捉摸。到洞察的精确位置和离子通道的功能特性以及它们在黑质神经元的树突的兴奋性和信息传输的作用之间的关系，直接树突录音是选择的方法。 本报告描述了可用于从黑质神经元的树突和相应的事后生物胞素标记获得单和双膜片钳记录的详细过程。用于修补体细胞和树突状膜的基本原理非常相似。实际上然而，从树枝状站点录音要求相比于体录音特定优化。成功的树突录音靠片，光学器件的优化调​​整，在记录的膜片吸管和稳定性的温和方法的质量。

<table><tbody><tr><td>Double-distillated water</td><td>Millipore</td><td>Super Q</td><td>Resistivity &gt;14 M&amp;Omega; cm, ideal 18.2 M&amp;Omega; cm at 25&amp;nbsp;&amp;deg;C, filtered (0.22 &amp;micro;m), https://www.merckmillipore.com/BE/en/product/Super-Q-Plus-Water-Purification-System,MM_NF-C1760</td></tr><tr><td>Microfilter candles</td><td>Robu</td><td>Porosity 3, 6 mm or 13 mm diameter</td><td></td></tr><tr><td>Tissue slicer with vibroprobe</td><td>Leica</td><td>VT1200</td><td>cutting parameters: speed 0.07, amplitude 1.40</td></tr><tr><td>Tissue slicer</td><td>Dosaka</td><td>DTK-1000</td><td>cutting parameters: speed 4, frequency 7</td></tr><tr><td>Razor blades</td><td>Gilette</td><td>Super Silver</td><td></td></tr><tr><td>Dissection tools</td><td>Braun, Aesculap</td><td></td><td></td></tr><tr><td>Reserve chamber</td><td></td><td></td><td>Custom-made</td></tr><tr><td>PP beckers</td><td>VWR</td><td>213-1725</td><td>400 ml</td></tr><tr><td>Syringe filter</td><td>Merk Millipore</td><td>Millex - GV 0.22 &amp;micro;m</td><td></td></tr><tr><td>Cyanoacrylate glue&amp;nbsp;</td><td>UHU</td><td>&quot;Sekunden Kleber&quot;</td><td>liquid glue</td></tr><tr><td>Recording chamber&amp;nbsp;</td><td>Luigs &amp;amp; Neumann</td><td>Slice mini chamber</td><td></td></tr><tr><td>Horizontal pipette puller</td><td>Sutter Instruments</td><td>Brown-Flaming P-97</td><td></td></tr><tr><td>Pipettes</td><td>Hilgenberg</td><td>1807524</td><td>2 mm o.d./1 mm i.d. glass capilaries, ends firepolished, washed</td></tr><tr><td>Dental wax</td><td>Colt&amp;egrave;ne/Whaledent</td><td></td><td>orthodontic tray wax strips</td></tr><tr><td>Platinum grid</td><td></td><td></td><td>to anchor slices in the recording chamber, custom made with a platinum disk</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-830</td><td>to fire-polish pipette tips</td></tr><tr><td>Potassium methyl sulfate</td><td>MP Biomedicals</td><td>215481</td><td></td></tr><tr><td>Biocytin</td><td>Molecular probes</td><td>B1592</td><td></td></tr><tr><td>Manometer</td><td>GREISINGER electronic</td><td>GDH 13 AN</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>FS</td><td></td></tr><tr><td>Dodt Gradient Contrast</td><td>Luigs &amp;amp; Neumann</td><td></td><td></td></tr><tr><td>Manipulators</td><td>Luigs &amp;amp; Neumann</td><td>LN Mini 25</td><td></td></tr><tr><td>Frame grabber</td><td>The Imaging Source</td><td>DFG/USB2pro</td><td></td></tr><tr><td>Camera</td><td>DAGE-MTI</td><td>NC-70</td><td></td></tr><tr><td>Condenser</td><td>Zeiss</td><td></td><td>high numerical aperture condenser working with oil</td></tr><tr><td>Objective</td><td>Zeiss</td><td>W Plan Apochromat 441470-9900 VIS-IR</td><td>63X magnification, long-distance, high numerical aperture 1.0 water-immersion objective</td></tr><tr><td>Fourfold-changer</td><td>Luigs &amp;amp; Neumann</td><td></td><td>between the camera and the microscope</td></tr><tr><td>Black &amp;amp; white video monitor</td><td>Sony</td><td>SSM-175CE</td><td>16-inch, 850 TV lines, analog</td></tr><tr><td>Sample Bottles, Amber Glass, with Cap</td><td>VWR</td><td>215-2409</td><td>to transfer slices from the setup to histology room</td></tr><tr><td>The Axon Guide</td><td>Molecular Devices</td><td></td><td>Book</td></tr><tr><td>Stimfit</td><td>https://github.com/neurodroid/stimfit</td><td></td><td>Analysis of electrophysiological data</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>441244</td><td>see SAFETY DATA SHEET and Dangerous Substances Directive (EU) before use</td></tr><tr><td>Well cell culture plate</td><td>Greiner-Bio-One</td><td>662160</td><td>for the staining of fixed slices</td></tr><tr><td>Triton X-100</td><td>Merk</td><td>108643</td><td>see SAFETY DATA SHEET and Dangerous Substances Directive (EU) before use</td></tr><tr><td>mouse anti-TH monoclonal primary</td><td>Immunostar</td><td>22941</td><td>working concentration: 1/1,000</td></tr><tr><td>goat anti-mouse secondary antibody - Alexa Fluor 568</td><td>Invitrogen - Thermo Fisher Scientific</td><td>A-11031</td><td>working concentration: 1/500</td></tr><tr><td>Normal goat serum</td><td>Dako</td><td>X0907</td><td></td></tr><tr><td>ProLong Diamond Antifade</td><td>Molecular Probes - Thermo Fisher Scientific</td><td>P36961</td><td></td></tr><tr><td>Fluorescein Avidin DCS</td><td>Vector Lab</td><td>A-2011</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>ImageJ</td><td>http://imagej.nih.gov/ij/</td><td></td><td></td></tr><tr><td>NeuronJ</td><td>http://www.imagescience.org/meijering/software/neuronj/</td><td></td><td>plugin for ImageJ</td></tr><tr><td>Imaris</td><td>http://www.bitplane.com</td><td></td><td>Reconstruction of biocytin filled neurons</td></tr><tr><td>Neuromantic</td><td>http://www.reading.ac.uk/neuromantic/</td><td></td><td>Reconstruction of biocytin filled neurons</td></tr><tr><td>WinWCP</td><td>http://spider.science.strath.ac.uk/sipbs/software_ses.htm</td><td></td><td>Analysis of electrophysiological data</td></tr></tbody></table>

subcellular patch-clamp recordings from the somatodendritic domain of nigral dopamine neurons

多巴胺能神经元树突接收和传达突触输入，支持动作电位反向传播和神经递质的释放。了解这些基本的功能将在这些神经元的信息传递线索。树突状膜片钳记录提供了可能直接检查树突和底层电压门控离子通道的电性能。然而，这些精细结构由于其小直径的不容易接触到的补丁吸管。这份报告描述了一步一步的过程来收集急性片多巴胺能神经元的树突稳定可靠的录音。电生理测量结合的细胞形态的事后恢复。成功的实验依靠改进的切片，解决方案和移液管，在与记录的结构相接触的光学元件和吸液管的稳定性的充分调整准备。索姆标准原则ATIC膜片钳记录被施加到树突但用吸管的温和的方法。这些多功能的技术可以被实现为处理有关枝晶的可激发性的各种问题。

上述协议打算促进使用树突膜片钳记录的数据的集合。这种技术与事后组织化学相结合，有助于深入了解许多电信号始发或蔓延到树突的机制。以树突直接访问补丁移液器是困难的，但特别注意的几个方法方面将提高录音的成功率。在稳定的体细胞记录足够的经验，实验者可以期望获得大多数尝试高电阻（GΩ）密封和完整的实验。一到两个高品质的树突状记录可以为每个夹层被收集。 含有健康的胞体和树突优质大脑切片的供应是访问这些精细结构（ 图1）的先决条件。的搜索标准一个用于选择这些神经元是平滑且均匀的表面遍布细胞，当与最佳调整光学观察到体细胞和树突低对比度（ 图1A和1B）。选择的神经元过强通常对比导致不稳定的记录（ 图1C和1C）。因此，例如神经元，应该避免。 相较于其他神经元，黑质多巴胺神经元显示特定的形态和电生理特性。这些通常与一个单个的体细胞移液管评估特征也可以在双体（ 图2）和somatodendritic记录（ 图3）观察到。龙超极化电流注入诱发称为"凹陷"（ 图2B和5D）膜电位整改。轴突起源，在大多数情况下，在一个树枝状站点识别使用互补标准13,25,26，这表明在这些神经元的树突隔室是多相（ 图3A - 3B）。其结果是，在AP首先观察到的隔室对应于其中轴突出现（ 图3C - 3D）的隔室13,25,26。轴突一般是由引起轴突的切割轴突疱切片64时终止，但没有系统检测到这种结构。 在黑质多巴胺神经元观察到连续的I H激活了明显下垂表明HCN亚基的高表达。为了确定I H突触信号的一体化的影响，产生并在somatodendritic双重录制55（&lt;通过树突状记录电极注入突触状电流（EPSC）波形STRONG&gt;图4）。这些模拟EPSCS的动力学进行了基于真实自然EPSCS的上升和下降时间常数。所得aEPSP记录与躯体电极。的I H阻滞剂ZD 7288的浴应用增加aEPSP的持续时间，指示我 h至突触信号（ 图4B）的时间过程的贡献。 ZD 7288还取消了膜电位整流确认从I H的激活（ 图4C）的凹陷的效果。用模拟EPSPS获得的结果可能是相关的使用电诱发EPSPS 65实验。映射的信道的在多巴胺神经元的somatodendritic域的精确分布，细胞贴附录音被执行（ 图5）。高电阻密封（&gt;&gt; 1GΩ）是绝对必要的细胞附着的录音（ 图5B </STRONG&gt;）。在多巴胺神经元的树突，I H与长极化电压步骤慢慢激活和当前稳态时达到数百毫秒（ 图5C）的后，如先前所示66。这一观察表明，在多巴胺神经元的树突表示HCN通道相比，那些在锥体神经元或其它类型的细胞10,16,66具有不同的亚单位。作为该信道的分配可能会在树突状隔室是不均匀的和作为树枝状隔室本身是异质的，枝晶（轴突载运或nonaxon轴承枝晶）的身份被IR-DGC图像与共焦图像进行比较发现后生物胞素染色（ 图3A和B）。细胞形态的恢复因此两个双重somatodendritic录音和用于经由后续体全细胞记录细胞贴附记录必要的。 图1. 可视化黑质多巴胺能神经元的胞体和树突近端用红外电视显微镜。 （A）IR-DGC一个黑质神经元DA显示索玛和近端树突和躯体都和树突状吸管的形象。双somatodendritic记录成功地在这个健康的神经元执行。观察低对比度和光滑表面遍布细胞的somatodendritic域。（B）一种健康的DA神经元的另一实例。（℃）DA神经元具有粗糙和不平坦的表面和更强的对比度。虽然已获得了双记录，稳定性是不理想的，并且记录时间为短（〜15分钟），由于在两个移液器接入电阻逐渐增加（D）示出的DA神经元的第二例的强烈对比的索玛和近端树突。在这种情况下，在全细胞模式突破后不久观察到的接入电阻的强劲增长。降低负压访问性的尝试不成功。在面板C和D的神经细胞可以在切片过程中已被破坏，并应避免用于实验。（E）在记录的开始的健康的DA神经元的同时双体记录。（F）的相同的神经元在图E与〜17分钟后，肿胀的细胞体。注意全没有相反，胞体的球一样的外观和大型核这不是健康的神经元明显的存在。 <a href="https://www.jove.com/files/ftp_upload/54601/54601fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54601/54601fig2.jpg" /> 图2。 </s仲&gt; 同时双体录音从神经元DA从一个神经元的DA双体录制过程中（A）IR-DGC形象。（B）超极化和去极化1秒长的电流注入（-160至80Pa，增加80 PA）和与两个补丁移液器同时记录对应的电压变化。请注意，凹陷在电压跟踪存在下与去极化电流脉冲在漫长的超极化电流步骤和AP后大后超极化。 <a href="https://www.jove.com/files/ftp_upload/54601/54601fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54601/54601fig3.jpg" /> 图3.双S IN DA的神经元omatodendritic记录。（A）一DA神经元的IR-DGC形象。树突和胞体之间的距离抽动吸在标有抗生物素蛋白偶联异硫氰酸荧光素（FITC）A组神经元的29微米。（B）共聚焦Z-投影图像。神经元在录制过程中充满了生物胞素。轴突源自接近由箭头指示的体细胞的近端枝晶。从记录在所述体躯（黑电压迹线）和树突面板A. AP中的神经元（C）的同时双somatodendritic全细胞电压记录（红色电压迹线）响应于经由所述体细胞（左一个1秒长的电流注入步骤;黑色电流曲线）和可替换的树突吸管（右;红色电流轨迹）（D）的体细胞和树突的AP在扩展时间标度所示。与任一体或树突电流注入，体细胞的AP（黑色）之前该树枝状的AP（红色）。在这个神经元的AP之间的延迟为120微秒和210微秒分别与体细胞和树突状电流注入。延误在AP的上升阶段于AP半数最大振幅进行测定。美联社在隔间接近其轴突出现，证实了以前的观察25,26首先观察。在这种情况下，树枝状记录从一个轴突缺乏树突制成。从轴突树突轴承是在文献记录的13（1在其图）的一个例子。 <a href="https://www.jove.com/files/ftp_upload/54601/54601fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54601/54601fig4.jpg" /> 图4. 人工EPSPS传播沿DA能神经元的Somatodendritic轴。 （A）一DA神经元的IR-DGC形象。树枝状和体细胞移液管之间的距离为45微米。两个补丁移液器在全细胞电流钳记录模式。（B）的电流在电流钳记录有树枝状吸管和用于生成人工EPSP（顶部）。通过树突记录吸管55的电流由一个EPSC状波形的注入得到的（;τ 衰变 = 3毫秒振幅= 100 pA的τ 上升 = 0.6毫秒）。在底部，繁殖记录在控制条件下（黑色）苏摩aEPSPs和I H受体阻滞剂ZD7288的浴后应用（30微米，红色曲线）。每个电压跟踪是40个单次扫描的平均值。观察ZD7288指示我 小时，以EPSPS的时间过程的贡献存在的EPSP持续时间大幅增加。（C）电压的控制条件下（黑色）索玛记录轨迹和30μMZD 7288后应用（红色）响应于一个长（30 PA; 1秒）超极化电流阶跃。请注意没有膜电位整流（SAG）堵塞后的I &lt;子&gt;小时。 <a href="https://www.jove.com/files/ftp_upload/54601/54601fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54601/54601fig5.jpg" /> 图 5. DA神经元树突 存在的 我 小时 。一个DA神经元和吸管近端枝晶（A）IR-DGC形象。（B）在细胞连接配置一个5 mV的电压指令期间电容和漏电流。密封电阻为5GΩ在本实施例（C）的超极化电压步骤;从一个膜电位的0毫伏诱导慢激活我 小时 （90毫伏顶部）。当前跟踪倒置，并配有一个单一的指数函数给出τ= 632毫秒的时间常数（D）电压以1秒hyper-全细胞记录，体细胞（图A）的反应和去极化电流脉冲（-160〜120 pA的，40 pA的增量）。体细胞全细胞记录在细胞附着在记录之后执行的。注意:如果没有接入点因TTX和Cd 2+的胞外应用细胞贴附记录期间抑制自发放电。这些电压门控钠离子和Ca添加2+电流阻滞剂来抑制动作电流。图A - D是来自同一神经元另一DA神经元和树突吸管（E）的IR-DIC图像由一个测试脉冲从一个50毫秒前脉冲在-120毫伏激活以0毫伏（F）的电压依赖性电流。在从面板大肠杆菌控股电位-80毫伏的神经元的近端树突切除一个外面向外补丁。请注意当前的时间依赖性失活。 <a href="https://www.jove.com/files/ftp_upload/54601/54601fig5large.jpg" target="_blank">请点击此处查看大图</a>版本这个数字。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>物质</td><td>克/摩尔</td><td>浓度</td><td>对于1升</td></tr><tr><td>氯化钠</td><td> 58.443 </td><td> 125毫米</td><td> 7.305克</td></tr><tr><td> 碳酸氢钠 </td><td> 84.007 </td><td> 25毫米</td><td> 2.100克</td></tr><tr><td>氯化钾</td><td> 74.551 </td><td> 2.5毫米</td><td> 0.186克</td></tr><tr><td>加入NaH 2 PO 4 </td><td> 137.99 </td><td> 1.25毫米</td><td> 0.172克</td></tr><tr><td>葡萄糖</td><td> 198.17 </td><td> 25毫米</td><td> 4.95克</td></tr><tr><td> 氯化镁 </td><td> 1 M（溶液） </td><td> 1毫米</td><td> 1毫升</td></tr><tr><td> 氯化钙 </td><td> 1 M（溶液） </td><td> 2毫米</td><td> 2毫升</td></tr><tr><td colspan="4">摩尔渗透压浓度:〜310 mOsmol / L（最佳范围:314 - 325 mOsmol / L），pH值= 7.4 </td></tr></tbody></table> 表1: 人工脑脊液（ACSF）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>物质</td><td>克/摩尔</td><td>浓度</td><td>对于1升</td></tr><tr><td>氯化钠</td><td> 58.443 </td><td> 87毫米</td><td> 5.084克</td></tr><tr><td> 碳酸氢钠 </td><td> 84.007 </td><td> 25毫米</td><td> 2.1001克</td></tr><tr><td>氯化钾</td><td> 7.551 </td><td> 2.5毫米</td><td> 0.18637克</td></tr><tr><td>加入NaH 2 PO 4 </td><td> 137.99 </td><td> 1.25毫米</td><td> 0.17248克</td></tr><tr><td> 氯化镁 </td><td> 1 M（溶液） </td><td> 7毫米</td><td> 7毫升</td></tr><tr><td>葡萄糖</td><td> 198.17 </td><td> 10毫</td><td> 1.9817克&lt;/ TD&gt; </tr><tr><td>蔗糖</td><td> 342.29 </td><td> 75毫米</td><td> 25.672克</td></tr><tr><td> 氯化钙 </td><td> 1 M（溶液） </td><td> 0.5毫米</td><td> 0.5毫升</td></tr><tr><td colspan="4">摩尔渗透压浓度:〜326 mOsmol / L，pH值= 7.4 </td></tr></tbody></table> 表2: 蔗糖ACSF以制备切片。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>物质</td><td>克/摩尔</td><td>浓度</td><td>为100毫升</td></tr><tr><td> KMeSO 4 </td><td> 150.2 </td><td> 120毫米</td><td> 1.8024克</td></tr><tr><td>氯化钾</td><td> 74.56 </td><td> 20毫米</td><td> 0.14912克</td></tr><tr><td> 氯化镁 </td><td> 1 M（溶液） </td><td> 2毫米</td><td> 200微升</td></tr><tr><td> 的 Na 2 ATP </td><td>五51.1 </td><td> 2毫米</td><td> 0.1102克</td></tr><tr><td> 的 Na 2 GTP </td><td> 523.2 </td><td> 0.5毫米</td><td> 0.02661克</td></tr><tr><td> 的 Na 2 -Phosphocreatine </td><td> 255.1 </td><td> 5毫米</td><td> 0.1275克</td></tr><tr><td> EGTA </td><td> 380.4 </td><td> 0.1毫米</td><td> 3.803毫克</td></tr><tr><td> HEPES </td><td> 238.31 </td><td> 10毫</td><td> 0.23831克</td></tr><tr><td>生物胞素</td><td> 1毫克/毫升</td><td></td><td> 0.1克</td></tr><tr><td colspan="4">摩尔渗透压浓度:〜302 mOsmol / L，pH值= 7.2用KOH调节</td></tr></tbody></table> 表 3: 双录音胞内溶液。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>物质</td><td>克/摩尔</td><td>浓度</td><td> 100毫升</td></tr><tr><td>氯化钾</td><td> 74.56 </td><td> 120毫米</td><td> 0.8947克</td></tr><tr><td> 氯化钙 </td><td> 1 M（溶液） </td><td> 2毫米</td><td> 200微升</td></tr><tr><td> 氯化镁 </td><td> 1 M（溶液） </td><td> 1毫米</td><td> 100微升</td></tr><tr><td> HEPES </td><td> 238.31 </td><td> 10毫</td><td> 0.23831 </td></tr><tr><td> TEA-CL </td><td> 165.7 </td><td> 20毫米</td><td> 0.3314克</td></tr><tr><td> 4-AP </td><td> 94.11 </td><td> 5毫米</td><td> 0.04705克</td></tr><tr><td>氯化钡2 </td><td> 244.26 </td><td> 1毫米</td><td> 0.02443克</td></tr><tr><td> 氯化镉</td><td> 183.32 </td><td> 0.02毫米</td><td> 0.3666毫克</td></tr><tr><td> TTX </td><td> 1毫米</td><td> 200纳米</td><td> 20微升</td></tr><tr><td colspan="4">摩尔渗透压浓度:〜290 mOsmol / L时，用D-葡萄糖（参考文献16）调节; pH值= 7.4 </td></tr></tbody></table> 表 4: 细胞贴附录音电极溶液。

Watch this Scientific Journal Video about Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons at JoVE.com

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

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 ...

subcellular-patch-clamp-recordings-from-somatodendritic-domain-nigral

Research

JoVE Journal

Neuroscience

14.9K 次观看.  Quartier Hôpital, 














University
of Liege. 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. 

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

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

膜片钳记录的成年大鼠完整背根神经节

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

科研

生物化学

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

一个NMDA受体电导的大鼠中脑多巴胺能神经元的动态钳技术中的应用

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

神经科学

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

在使用单细胞RT - PCR技术的黑质神经元电压门控钾通道mRNA表达分析

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

单细胞的多巴胺释放，同时电压钳和电流分析法测量

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

膜片钳后单细胞多重反转录聚合酶链反应

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

One-channel Cell-attached Patch-clamp Recording

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

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

单通道细胞贴附膜片钳记录

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

生物学

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

从新生儿啮齿动物中分离和培养前庭和螺旋神经节 Somata 以进行膜片钳记录

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Patch-Clamp Recordings from the Dendrite of a Dopaminergic Neuron in a Brain Slice

Source: Engel, D. <a href="https://app.jove.com/v/54601">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for patch-clamp recording from the dendritic membrane surface. This helps to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels in dopaminergic neurons.

来自脑切片中多巴胺能神经元树突的膜片钳记录

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

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

Source: Iyer, M. R., et al. <a href="https://app.jove.com/v/64908">Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.</a> J. Vis. Exp. (2023).
This video demonstrates the patch-clamp technique for measuring ionic currents in inner ear sensory neuron cell bodies. It details the preparation and use of a polished patch-clamp pipette filled with an internal solution containing amphotericin B, which perforates the membrane to allow ion movement while preserving intracellular conditions. Voltage pulses are applied to trigger and record ion channel activity, providing insights into their properties and behavior.

内耳感觉神经元细胞体的穿孔膜片钳记录

1. Recording
NOTE: In this procedure, patch-clamp recordings from the isolated ganglion are typically performed 12-24 h after the plating. Other labs have ...

Imaging and Optical Techniques

Whole-Cell Patch Clamp Recording in a Substantia Gelatinosa Neuron of a Spinal Cord Slice

Source: Zhu, M., et al. <a href="https://app.jove.com/v/58479">Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for performing electrophysiological recordings in substantia gelatinosa (SG) neurons of spinal cord slices, using whole-cell patch-clamp techniques to analyze neuron firing patterns. This method allows the study of neural responses to stimuli in SG neurons of the spinal cord.