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 border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="137">
			<td height="137" style="height:138px;width:156px;">Double-distillated water</td>
			<td style="width:143px;">Millipore</td>
			<td style="width:137px;">Super Q</td>
			<td style="width:187px;">&#160;Resistivity &#62;14 M&#937; cm, ideal 18.2 M&#937; cm at 25&#176;C, filtered (0.22 &#181;m), https://www.merckmillipore.com/BE/en/product/Super-Q-Plus-Water-Purification-System,MM_NF-C1760</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">Microfilter candles</td>
			<td style="width:143px;">Robu</td>
			<td style="width:137px;">Porosity 3, 6 mm or 13 mm diameter</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:156px;">Tissue slicer with vibroprobe</td>
			<td style="width:143px;">Leica</td>
			<td style="width:137px;">VT1200</td>
			<td style="width:187px;">cutting parameters: speed 0.07, amplitude 1.40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">Tissue slicer</td>
			<td style="width:143px;">Dosaka</td>
			<td style="width:137px;">DTK-1000</td>
			<td style="width:187px;">cutting parameters: speed 4, frequency 7</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Razor blades</td>
			<td style="width:143px;">Gilette</td>
			<td style="width:137px;">Super Silver</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Dissection tools</td>
			<td style="width:143px;">Braun, Aesculap</td>
			<td style="width:137px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Reserve chamber</td>
			<td style="width:143px;"></td>
			<td style="width:137px;"></td>
			<td style="width:187px;">Custom-made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">PP beckers</td>
			<td style="width:143px;">VWR</td>
			<td style="width:137px;">213-1725</td>
			<td style="width:187px;">400 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Syringe filter</td>
			<td style="width:143px;">Merk Millipore</td>
			<td style="width:137px;">Millex - GV 0,22 &#181;m</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Cyanoacrylate glue&#160;</td>
			<td style="width:143px;">UHU</td>
			<td style="width:137px;">&#34;Sekunden Kleber&#34;</td>
			<td style="width:187px;">liquid glue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Recording chamber&#160;</td>
			<td style="width:143px;">Luigs &#38; Neumann</td>
			<td style="width:137px;">Slice mini chamber</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">Horizontal pipette puller</td>
			<td style="width:143px;">Sutter Instruments</td>
			<td style="width:137px;">Brown-Flaming P-97</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">Pipettes</td>
			<td style="width:143px;">Hilgenberg</td>
			<td style="width:137px;">1807524</td>
			<td style="width:187px;">2 mm o.d./1 mm i.d. glass capilaries, ends firepolished, washed</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Dental wax</td>
			<td style="width:143px;">Colt&#232;ne/Whaledent</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">orthodontic tray wax strips</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">Platinum grid</td>
			<td style="width:143px;"></td>
			<td style="width:137px;"></td>
			<td style="width:187px;">to anchor slices in the recording chamber, custom made with a platinum disk</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Microforge</td>
			<td style="width:143px;">Narishige</td>
			<td style="width:137px;">MF-830</td>
			<td style="width:187px;">to fire-polish pipette tips</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:156px;">Potassium methyl sulfate</td>
			<td style="width:143px;">MP Biomedicals</td>
			<td style="width:137px;">215481</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:156px;">Biocytin</td>
			<td style="width:143px;">Molecular probes</td>
			<td style="width:137px;">B1592</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:156px;">Manometer</td>
			<td style="width:143px;">GREISINGER electronic</td>
			<td style="width:137px;">GDH 13 AN</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:156px;">Microscope</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:137px;">FS</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:156px;">Dodt Gradient Contrast</td>
			<td style="width:143px;">Luigs &#38; Neumann</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Manipulators</td>
			<td style="width:143px;">Luigs &#38; Neumann</td>
			<td style="width:137px;">LN Mini 25</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:156px;">Frame grabber</td>
			<td style="width:143px;">The Imaging Source</td>
			<td style="width:137px;">DFG/USB2pro</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Camera</td>
			<td style="width:143px;">DAGE-MTI</td>
			<td style="width:137px;">NC-70</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:46px;width:156px;">Condenser</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">high numerical aperture condenser working with oil</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:156px;">Objective</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:137px;">W Plan Apochromat 441470-9900 VIS-IR</td>
			<td style="width:187px;">63x magnification, long-distance, high numerical aperture 1.0 water-immersion objective&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:156px;">Fourfold-changer</td>
			<td style="width:143px;">Luigs &#38; Neumann</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">between the camera and the microscope</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:156px;">Black &#38; white video monitor</td>
			<td style="width:143px;">Sony</td>
			<td style="width:137px;">SSM-175CE</td>
			<td style="width:187px;">16-inch, 850 TV lines, analog</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:156px;">Sample Bottles, Amber Glass, with Cap</td>
			<td style="width:143px;">VWR</td>
			<td style="width:137px;">215-2409</td>
			<td style="width:187px;">to transfer slices from the setup to histology room</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">The Axon Guide</td>
			<td style="width:143px;">Molecular Devices</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">Book</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:46px;width:156px;">Stimfit</td>
			<td style="width:143px;">https://github.com/neurodroid/stimfit</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">Analysis of electrophysiological data</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:156px;">Paraformaldehyde</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:137px;">441244</td>
			<td style="width:187px;">see SAFETY DATA SHEET and Dangerous Substances Directive (EU) before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:156px;">Well cell culture plate</td>
			<td style="width:143px;">Greiner-Bio-One</td>
			<td style="width:137px;">662160</td>
			<td style="width:187px;">for the staining of fixed slices</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">Triton X-100</td>
			<td style="width:143px;">Merk</td>
			<td style="width:137px;">108643</td>
			<td style="width:187px;">see SAFETY DATA SHEET and Dangerous Substances Directive (EU) before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">mouse anti-TH monoclonal primary</td>
			<td style="width:143px;">Immunostar</td>
			<td style="width:137px;">22941</td>
			<td style="width:187px;">working concentration: 1/1000</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">goat anti-mouse secondary antibody - Alexa Fluor 568</td>
			<td style="width:143px;">Invitrogen - Thermo Fisher Scientific</td>
			<td style="width:137px;">A-11031</td>
			<td style="width:187px;">working concentration: 1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Normal goat serum</td>
			<td style="width:143px;">Dako</td>
			<td style="width:137px;">X0907</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">ProLong Diamond Antifade</td>
			<td style="width:143px;">Molecular Probes - Thermo Fisher Scientific</td>
			<td style="width:137px;">P36961</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Fluorescein Avidin DCS</td>
			<td style="width:143px;">Vector Lab</td>
			<td style="width:137px;">A-2011</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:156px;">Confocal microscope</td>
			<td style="width:143px;">Olympus</td>
			<td style="width:137px;">FV1000</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:45px;width:156px;">ImageJ</td>
			<td style="width:143px;">http://imagej.nih.gov/ij/</td>
			<td style="width:137px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:156px;">NeuronJ</td>
			<td style="width:143px;">http://www.imagescience.org/meijering/software/neuronj/</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">plugin for ImageJ</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">Imaris</td>
			<td style="width:143px;">http://www.bitplane.com</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">Reconstruction of biocytin filled neurons</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:156px;">Neuromantic</td>
			<td style="width:143px;">http://www.reading.ac.uk/neuromantic/</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">Reconstruction of biocytin filled neurons</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:61px;width:156px;">WinWCP</td>
			<td style="width:143px;">http://spider.science.strath.ac.uk/sipbs/software_ses.htm</td>
			<td style="width:137px;"></td>
			<td style="width:187px;">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

14.8K 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 from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</subATP酶微囊的制备和表演膜片钳记录的Submitochondrial泡膜技术

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

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 ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

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