Name: intracellular recording, sensory field mapping, and culturing identified neurons in the leech, hirudo medicinalis
Uploaded: 2013-11-04T21:00:00.000+00:00
Duration: 11 min 53 s
Description: Watch this Scientific Journal Video about Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis at JoVE.com

1。电极和盐溶液的制备<ol><li>准备生理盐水和电极将动物解剖前。水蛭林格氏液由以下部分组成：115.3毫米氯化钠，1.8毫米氯化钙 ，4.0毫米氯化钾，10毫米的Tris /马来酸或HEPES。将pH调节至7.4。 </li><li>浸清洁的银线放入少量的漂白剂20分钟或直到丝具有光滑的暗灰色涂层准备的Ag-CL线。使用前用蒸馏水洗丝。然后将导线放置成可插入放大器微量持有者。 </li><li>用吸管拉马准备硼硅玻璃的锋利玻璃电极。电极电阻应为20兆欧姆量级。 </li><li>回填移液器用3M乙酸钾溶液，并插入在插入微量支架。 </li><li>对于一个细胞外电极制备如上所述尖锐的玻璃吸管，打破尖端背面由揉附近的锋利边缘再次出现大幅吸管。那么火抛光尖，以便它不会撕裂连接词。塑料外电极也只要是在电极和神经之间的良好接触工作。 </li><li>插入外记录电极插入微量支架（与银氯导线），它配有一橡胶管和注射器进行抽吸。用注射器抽取盐水进入电极至少在银线的电平。 </li><li>通过遵循本手册为您的特定信号记录器的设置硬件（放大器和刺激隔离器）和软件记录参数。见Leksrisawat 等人 22可以看到在下面描述的实验中使用的设备的细节。 </li></ol> 2。腹神经节解剖<ol><li>解剖水蛭在一个大的有机硅弹性体内衬菜水蛭林格氏液。如果动物被纵向拉伸会更容易除去腹神经索（ 见图1A）。 </li><li>与＃10或＃11的大小手术刀，作一纵切口在腹部中线很浅的切口。关于腹侧切口的中间2/3rd示于图1B中 。尽量不要通过黑色长筒丝袜（腹血窦）切，直到动物被完全打开，血窦被拉伸而不扭结。这血窦运行动物沿腹中线的长度。 VNC的包含在此的血管内。 </li><li>后皮肤已被切断动物的全长用细光圈剪刀缺口的血窦（ 图2）。滑的窦下的细虹膜剪一个叶片和稍微提高刀片将其从神经组织中分离出来。切窦VNC的长度，同时避免了神经根，神经节段或神经节之间的连接词。 </li><li>接着，取出腹神经索的一部分远端切到血管连接根部的根。沿腹神经索的长度每节重复此过程。 </li><li>的一个或两个神经节段转移至一个较小的培养皿中。神经节或神经节单需固定绷紧有些呆滞。 </li><li>针的连接词和根部，因此神经节神经胶质鞘稍微拉伸，以防止神经元细胞体通过所述胶质鞘并进入感兴趣的神经元刺穿时走动。该引脚应放在通过黑色窦的根部，两个联结的中间，尽量减少轴突损伤（ 图3）。 </li><li>将培养皿用制剂，在显微镜下，用蜡将其固定在该盘，以防止移动的底部。 </li><li>利用反射镜和聚光看到的神经元， 如图4A小心聚焦光束（示意于图4B中所示</st荣&gt;）。注意两个大Retzius细胞在神经节的中心。 </li><li>使RZ，T，P和N个神经元的细胞内记录时，小心地将电极的尖端在感兴趣的细胞和轻轻降低前端直到一个凹坑被认为是在胶质鞘。 </li><li>点击电极支架或机械手（非常仔细），使电极尖端“啪啪”到感兴趣的细胞。 </li><li>注入正向电流（1-10 NA）的短脉冲（30-40毫秒）进入细胞内，以唤起动作电位。 </li></ol> 3。从腹神经节准备培养神经元<ol><li>脚的神经节腹侧中含有L-15与胎牛血清（FCS）（ 图3）干净的菜。脚根，使神经节稍微拉紧作为细胞内的录音做以上。用细剪刀，尼克胶质囊的一端，滑1剪刀刀片包膜下，然后穿过胶质囊切如图5A。 </li><li>加胶原酶/分散酶的100微升等分试样，以在振荡器上15分钟，在烹调和地点。通过使外露的细胞体周围的培养基中检查细胞。返回的单元格的摇床另外15分钟或更长的时间，直到细胞出来以最小的吸力。 </li><li>使用附连到注射器从神经节轻轻提取细胞的移液管。火抛光该移液管尖端，使得它比细胞体直径稍大。对于RZ细胞在成年水蛭，直径应为约50微米。 </li><li>准备了各种移液器具有不同直径的无灯丝，因为这降低了细胞粘附吸管内的机会。存储移液器在含80％乙醇（EtOH中），使用棉球在底部，以保护技巧从碎裂的瓶子。记住吸细胞前具有中等洗出乙醇吸管。 </li><li>酶处理后，洗出的含酶介质与L-15（有FCS）2倍。找出感兴趣的细胞，轻轻拉动注射器（ 图6A）吸引他们到吸管。放电吸气细胞进入另一道菜，其中包含L-15（带FCS）。 </li><li>到现在为止，所有的程序都是非无菌。执行在无菌罩下面的步骤。将3-4滴大滴无菌的L-15（与FCS）在无菌培养皿内的不同位置。 </li><li>吸取细胞进入前面提到滴之一，在下拉周围吹他们把它们洗干净。在3或4滴介质的重复这个串联。 </li><li>把细胞在一个干净的培养皿中充满了L-15（有FCS）。 </li><li>于室温孵育1-24小时，在室温下进行。事后清洁单元可电镀。将其留在细胞几个小时或隔夜外基质容易脱落。立即板上的细胞来产生较长的过程。 </li><li>使用无菌基材涂层微孔二<html> sh和允许它培养实验前彻底晾干（过夜）。 </li><li>后盘已经涂，用无菌水轻轻冲洗培养皿，然后用少量的L-15培养基中。请记住，使用L-15解决方案，而在FCS加入使细胞附着在菜。该介质可以被轻轻地改变到L-15与FCS后的细胞粘附在基材上。如果细胞要使用镀覆，然后切换到中的L-15与FCS后几个小时是没有必要的。 </li><li>定位单元彼此相邻于镀的时间以诱导快速突触（ 图6B）。后在神经元中的配对：24小时测量的生理活性。 </li><li>利用细胞内电生理技术，以培养对细胞的记录RP或突触反应（1刺激通过神经元从配对的细胞的细胞内电极和记录反应使用的是第二个胞内电极）。 </li></ol>标题“&gt; 4。神经节体墙准备<ol><li>针在如上所述的弹性体衬里的培养皿水蛭背面向下。 </li><li>针在皮肤一侧上被切断的神经节的另一侧根部（ 图7A，放大在图7B），或使一个窗口上的水蛭超过一个神经节（ 图7C）与所有的根到身体的腹侧墙壁完好无损。 </li><li>后获得稳定的细胞内记录中的感觉细胞（T，P，N）的使用已经火抛光，轻轻触碰在同一网段的皮肤小玻璃棒之一。如果从正被记录的神经元是N细胞，然后更多的压力将需要被施加到皮肤上。在大多数情况下，如果皮肤被夹用镊子N个小区将只响应。因为这个原因，这些细胞应当最后尝试因为人们可能损伤皮肤或通过干扰制剂以这样的方式置换细胞内重新编码。 </li><li>每个人都应该能够快速地绘制皮肤和神经节与细节足以说的其中一个触摸可用于确定特定的神经元的感受域的地图。从尽可能多的T和P细胞可能在一节两侧录制完毕后试了N细胞。 </li><li>为了检验是否感觉神经元有通过在延髓或尾的方式，然后在相邻的段皮肤根部的连接词旅行过程中，神经节之间的连接词可切割和感受野复查中问候检测的蔓延。基本上，一个是检查是否有一直在感受野的任何损失。 </li></ol> 5。注射荧光染料<ol><li>准备如上述那样水蛭的神经节。 </li><li>填充微电极与荧光黄-CH（3.0％）和氯化锂（300毫摩尔）的溶液中。 </li><li>如上所述刺穿的神经节的腹侧感觉细胞或RZ小区之一以上。 </li><li>通过使负电流（3nA的）经过15分钟的电极注入的染料进入细胞。设置脉冲的持续时间为100毫秒，并且频率为2赫兹。 </li><li>通过激励染料与水银灯和FITC / GFP滤波器组可视化的填充染料的神经元。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+RNAi+and+ectopic+gene+expression+in+the+medicinal+leech.">Localized RNAi and ectopic gene expression in the medicinal leech.</a> J. Vis. Exp. (14), e697 (2008).</li><li>Firme, C. P., Natan, R. G., Yazdani, N., Macagno, E. R., Baker, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+expression+of+select+innexins+in+individual+central+neurons+couples+them+to+pre-existing+neuronal+or+glial+networks+that+express+the+same+innexin.">Ectopic expression of select innexins in individual central neurons couples them to pre-existing neuronal or glial networks that express the same innexin.</a> J. Neurosci. 32, 14265-14270 (2012).</li><li>Siddall, M. E., Trontelj, P., Utevsky, S. Y., Nkamany, M., Macdonald, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+molecular+data+demonstrate+that+commercially+available+medicinal+leeches+are+not+Hirudo+medicinalis.">Diverse molecular data demonstrate that commercially available medicinal leeches are not Hirudo medicinalis.</a> Proc. Biol. Sci. 274, 1481-1487 (2007).</li></ol>

作者什么都没有透露。

这组实验的目的是1）从识别的神经元教，并表现出细胞内记录的基本原则和2）认识到的电信号，可以产生用成年水蛭这些特定类型的神经元的特征形状。有调查采用水蛭的神经生理学研究丰富的历史和研究工作正在进行中处理的神经回路，并在突触的修复和再生10在开发过程中，以及基因调控的问题。神经调节，特别是通过生物胺的途径，也于水蛭是一个成功的研究方向。通过盐溶液加入多巴胺等药物制剂的神经系统，它已经表明，多巴胺调节参与决策23和运动模式的爬行行为24,25神经网络。这种实验方法是简体乐和廉价的方式来把调制成教学实验室，并调节盐溶液的离子构成是教导能斯特和戈德曼霍奇金 - 卡茨方程的有效方法。 为了取得成功与神经节筹备工作有神经节之前，试图戳躯体与细胞内电极拉紧是非常重要的。否则，在神经胶质包推下来时，躯体会移动。此外，desheathing神经节用于培养神经元中除去时，在整个神经节切割时所以胞体不被损坏，因为它们将被删除避免感兴趣区域。此外，过度的温育中的酶可导致神经元太脆弱被删除。它需要一些试验和错误，以达到理想的孵化时间，因为每批酶略有不同在他们的行动。 进一步的调查，以优化培养基可以改善这个技术。变量项因素的影响，如胰岛素，糖，或生长因子的存在下，可以检查对更长的维护文化。以确定是否细胞保持它们的合成发射机，如五羟色胺的合成在RZ细胞的能力，中性红染色可以应用于标记的血清素的过程。还小胶质细胞浸润至受伤和再生组织可以通过核污渍日受伤后进行量化。 虽然这种制剂有很多好处，提供在研究再生与修复，以及神经系统的电路，在突触的药理学和分子鉴定的各种受体亚型还没有得到充分的特点。一个表达序列标签库可用26和医用水蛭基因组中，目前正在组装27，但在水蛭的主要限制相比，一些模型制剂是修改的基因组中选择性基因在特定细胞类型的能力。在胚胎吨他的限制已经解决了注射DNA或双链RNA来调节基因表达28。基因枪技术最近被用于表达无脊椎动物连接蛋白（innexin）的蛋白质在神经元的水蛭。从水蛭verbana（HVE-inx6）的innexin的表情足以异位间隙连接的医用水蛭29的形成。医用水蛭-或H。马鞭草因为它是30是一定要推进我们的生理和电突触的演变，在未来几年的理解。

水蛭制剂是研究识别（感觉，运动和动物的中枢神经系统（CNS）内跨神经元的功能非常有用。水蛭神经元的有用的特性是它们的动作电位和生物物理特性的形状特征的给定的细胞类型（ 例如 P，N，T，RZ）。另外的神经元可以从在适当的培养基中，将细胞保持它们的电特性进行了长达45天的1,2动物和培养被删除。 水蛭神经节学习如何做电生理记录的一个显着优点是细胞特征性和可靠地安排在一个模式，可以让神经节识别不同类型的细胞从神经节神经节，并从动物到动物。独特的地理位置和水蛭神经节神经元的形态进行注意到Retzius早在1891年3。知识次方ë身份和一个神经元的功能是有利的侦查神经回路和用于制备实验用给定的细胞类型。由于神经节它们与解剖显微镜可见，与胞体可与微电极，用于记录它们的电学性能被选择性地刺穿内神经元的相对大的胞体。 染料分子和大分子可注入单个细胞，并研究通过膜片钳信道特性。离子电流，在各种神经元的特征动作电位的各种形状产生已确定4-7。从神经支配的格局和分支中枢神经系统内单个神经元的调查，突触连接已经转载于文化与鉴定神经元2,8。研究中的水蛭神经节提供了洞察功能电路的开发，可以与电测量以及与wholË动物行为的研究。水蛭中枢神经系统还曾担任调查涉及神经元的修复和再生，在整个动物和文化4-6,9-12机制的一个重要前提。 在哺乳动物的大脑是一个艰巨的任务，在属性和个人识别神经元的连接方面解释的行为。原则上我们可以希望，不太复杂的系统，如水蛭的研究可能有助于确定在高等动物中使用的基本机制。所使用的背后游泳图案13,14和15心脏节律的连接得到了很好的特征水蛭。一些水蛭的神经元形成电气和化学通讯的能力是耐人寻味。有些连接是双向的，而有些则是单向的化学性质，但双向电7。了解动物中的突触连接，以及重新创建相同的典型E在培养皿中的连接是调查突触16-18以及19突触药理分析的强大工具。这里所描述的细胞内记录和刺激技术提供了药理实验和研究神经行为学和发展的基础。 此外，该段腹神经索可解剖与神经支配的皮肤补丁。以保持贴剂的皮肤和神经元存活的能力，感官感受域可以通过从在CNS各自腹腔神经节内的感觉神经元的细胞体（ 即胞体）记录的电信号被探测。从而可以通过应用不同程度的力在皮肤上评估感觉神经末梢的敏感性和地图感受野：轻轻一碰，压力和有毒（痛苦的）刺激20,21。 这水蛭神经节皮制剂的优点是，一个CAÑ​​画解剖感受野地图和跟踪神经元的过程来确定的神经元一旦电反应被记录。下述三种水蛭的实验都可以完成多次使用一个单一的标本，这使得这是一个成本有效的教学模块教学实验室。

<table><tbody><tr><td>Sylgard</td><td>Dow Corning</td><td>182 silicone kit</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>Tris/maleic acid</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Bleach&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td>To chloride silver wire</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>221465</td><td>To adjust pH</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>H1758</td><td>To adjust pH</td></tr><tr><td>Collagenase/Dispase</td><td>Boehringer Mannheim</td><td>269-638</td><td></td></tr><tr><td>Leibovitz L-15 with L-Glutamine</td><td>Gibco</td><td>041-01415H</td><td></td></tr><tr><td>Fetal calf serum</td><td>Ready Systems Ag</td><td></td><td></td></tr><tr><td>04-001</td><td></td><td></td><td></td></tr><tr><td>D-Glucose-Monohydrate</td><td>Merck</td><td>8342</td><td></td></tr><tr><td>Gentamycin sulfate&amp;nbsp;</td><td>any supplier</td><td></td><td>10 mg/ml</td></tr><tr><td>Glutamine</td><td>Gibco</td><td>043-0503H</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>3375</td><td>Free acid, crystalline</td></tr><tr><td>Concanavalin A Type IV</td><td>Sigma</td><td>C 2010</td><td></td></tr><tr><td>Poly-L-Lysine Hydrobromide</td><td>Sigma</td><td>P 7890</td><td>MW 15-30,000</td></tr><tr><td>&amp;nbsp;Microwell plate, 60 x 10 &amp;mu;l</td><td>Falcon</td><td>3034</td><td>Flat bottom, 10 in a bag</td></tr><tr><td>Dissecting tools</td><td>World Precision Instruments</td><td>assortment</td><td></td></tr><tr><td>Intracellular electrode probe</td><td></td><td></td><td></td></tr><tr><td>Faraday cage</td><td></td><td></td><td></td></tr><tr><td>Insect Pins</td><td>Fine Science Tools, Inc</td><td>26001-60</td><td></td></tr><tr><td>Dissecting microscope (100X)</td><td></td><td></td><td></td></tr><tr><td>Fiber optic lamp</td><td></td><td></td><td></td></tr><tr><td>Small adjustable mirror</td><td></td><td></td><td>To direct light beam toward the preparation.</td></tr><tr><td>Glass electrodes</td><td>Sigma-Aldrich</td><td>CLS7095B5X</td><td>Box of 200, Suction electrodes</td></tr><tr><td>Micromanipulator</td><td>World Precision Instruments</td><td>MD4-M3-R</td><td>&amp;nbsp;Can fix for base or on a metal rod</td></tr><tr><td>Raised preparation stand</td><td></td><td></td><td></td></tr><tr><td>Silver wire (10/1,000 inch)</td><td>A-M Systems</td><td>782500</td><td></td></tr><tr><td>Computer</td><td>any company</td><td></td><td></td></tr><tr><td>AC/DC differential amplifier</td><td>A-M Systems</td><td>Model 3000</td><td></td></tr><tr><td>PowerLab 26T</td><td><a href="http://www.adinstruments.com/" target="_blank"><img src="/img/new_window.png"> AD Instruments</a></td><td>27T</td><td></td></tr><tr><td>Head stage</td><td><a href="http://www.adinstruments.com/" target="_blank"><img src="/img/new_window.png"> AD Instruments</a></td><td></td><td>Comes with AC/DC amplifier</td></tr><tr><td>LabChart7</td><td><a href="http://www.adinstruments.com/" target="_blank"><img src="/img/new_window.png"> AD Instruments</a></td><td></td><td></td></tr><tr><td>Electrical leads</td><td>any company</td><td></td><td></td></tr><tr><td>Glass tools&amp;nbsp;</td><td>make yourself</td><td></td><td>For manipulating nerves</td></tr><tr><td>Cable and connectors</td><td>any company</td><td></td><td></td></tr><tr><td>Pipettes with bulbs&amp;nbsp;</td><td>Fisher Scientific</td><td>13-711-7</td><td>Box of 500</td></tr><tr><td>Beakers</td><td>any company</td><td></td><td></td></tr><tr><td>Wax or modeling clay</td><td>any company or local stores</td><td></td><td></td></tr><tr><td>Stimulator</td><td>Grass Instruments</td><td>SD9 or S88</td><td></td></tr><tr><td>Plastic tip for suction electrode</td><td>local hardware store (Watt&amp;rsquo;s brand)</td><td>&amp;frac14; inch OD x 0.170 inch ID</td><td>Cut in small pieces. Pull out over a flame and cut back the tip to the correct size.</td></tr></tbody></table>

intracellular recording, sensory field mapping, and culturing identified neurons in the leech, hirudo medicinalis

淡水水蛭， 医用水蛭 ，是一个已经被用来解决在神经生理学，神经行为学和发育生物学等领域的科学问题一个多才多艺的模式生物。本报告的目的是为了巩固从水蛭系统实验技术成一个单一的文章，将用来生理学家具有专长的其他神经系统制剂，或生物学的学生很少或根本没有电的经验。我们将演示如何解剖水蛭来自于神经节确定的神经回路在细胞内记录。接下来，我们显示如何已知功能的单个细胞可从神经节在培养皿中进行培养，以及如何从在培养这些神经元来记录被删除。然后，我们演示了如何准备支配的皮肤被用于映射感觉或运动领域的一个补丁。这些水蛭制剂仍然被广泛使用，以解决神经网络结构的基本电性能KS，行为，突触和发展。他们还对神经科学和生理学教学实验室适当的培训模块。

来自水蛭的神经节感觉神经元的细胞内记录的特征是在图4C所示的不同的电压-时间的痕迹。在健康水蛭的神经节细胞的静息细胞的膜电位为-45至-60毫伏。如果电势更小（少负）1应改变玻璃电极，或者如果潜在仍然很低，在移动到神经索的另一部分。自发动作电位的小幅度可能表明该电极是在运动神经元。如果这种情况发生在神经节体壁中的电流注入可以被用来确定该神经支配任何仍附着在皮肤上的肌肉。纤维环竖运动神经元（AE）是在神经节（ 图4B）的腹侧，它会导致年轮（脊或段）通过激活环竖肌上升。 注入电流的感觉细胞，应引起动作电位s的图4C中所示的特性的波形。如果波形看起来不同，它可能是由于不平衡电桥或抵消电容补偿。每个人都应该咨询，伴随着信号采集单元为如何平衡电桥在细胞内详细的说明书。通知的刺激伪像在图4C中的电流脉冲的开始和结束。这是神器怎么看时，电桥平衡。 神经元由神经节水蛭原代培养形成电和化学突触的菜。最初培养的细胞的电特性是不完全，因为它们在体内是相同的。然而，几天后的冲动几乎无法区分这些记录在神经节。这表现在图6C一对​​RZ细胞。 7小时后在文化突触电位的振幅为1〜2毫伏。经过56小时培养SYnaptic势比10毫伏（中上部的曲线图6C）放大。动作电位的形状56hr在培养后也发生变化。根据所描述的条件下，这些细胞是可行的几天。 细胞间通讯可以通过刺激神经根或连接词和从神经节神经元记录来证明。刺激这些神经会产生强大的突触电位和动作电位在不同的神经节细胞。在图8B痕迹从Retzius细胞已被从动物身上取出，并在培养皿中寄托了神经节记录。黑色的痕迹被诱发的应用与细胞外电极的结缔组织阈下刺激。增加刺激电压引起细胞触发一个动作电位（红色曲线）。荧光染料或辣根过氧化物酶可以被注射到这些细胞中，研究它们的形态。荧光黄注射到一个RZ神经元通过使负电流通过电极。另一种方法是利用空气压力的抽吸。 图9A示出在注射开始后不久的染料的染料注入。三十分钟后，将染料已经已经扩散到神经根（ 图9B）。染料的类型取决于应用程序， 如小分子量的染料，可通过间隙连接来识别电突触。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig1.jpg" width="600px" /> 图1。 。腹水蛭清扫术（一）动物被彻底固定腹侧于弹性体内衬菜充满了水蛭生理盐水（B）浅切口跨越前：后轴刚才外侧中线，以避免损坏神经线。黑色的血窦（其中含有多种NS腹神经索）可以很容易地看到。请注意，一个暴露神经节相比，鞘是白色的。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig2.jpg" width="600px" /> 图2：黑色窦夹层的血管（箭头）应仔细切开一侧，露出腹神经索（双箭头）。离开血管段周围的节段性根部用作手柄解剖和记录菜肴之间传输时，操纵腹神经索完好。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3"FO：内容宽度=“3英寸”SRC =“/ files/ftp_upload/50631/50631fig3.jpg”宽度=“400像素”/&gt; 腹神经索的图3。隔离部分由其余的根段血管片段，并在段连接词的两端钢钉腹神经索暴露了神经节的电生理记录。绷紧拉伸神经节使人们看到了可识别的细胞体，将允许用锋利的玻璃电极的胶质鞘更容易渗透。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig4.jpg" width="600px" /> 图4。水蛭神经节（一）理想情况下，Retzius细胞（R）和其他大细胞体（P-细胞按解剖。URE，T型触摸，N-伤害，AE-环竖）将清晰可见，如果在显微镜和聚光设置正确（B）细胞在神经节的示意图。会发生在每个神经节中的胞体位置的微小差异是由于神经节是如何拉伸和固定的拉紧的，从这些神经元的胞 ​​体记录的（C）的动作电位具有特性的波形。这些尖峰是由注入正电流进入细胞（方波脉冲叠加的电压-时间的痕迹）诱发。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig4highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig5.jpg" width="600px" /> 图5。Desheathing神经节删除单个的细胞体（A）的胶质鞘应在整个神经节被精心切割，以避免利益损害细胞。红线表示切，以避免击中RZ神经元的胞体。打开外壳给人的消化酶接触到结缔组织基质（B）健康细胞体鼓胀出来的胶质鞘已被打开后，神经节中。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig5highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig6.jpg" width="600px" /> 图6：从培养水蛭神经节神经元（A）细胞从神经节使用微量吸并转移至含有培养基一道菜。（b）将多个单元彼此接近会增加突触的识别神经元之间的比率。在这里，STU两个神经元的国会议员在文化表现出略微不同的电性能形成突触（C）神经元。顶部迹线示出通过刺激相邻小区7小时和56小时的细胞铺板后诱发的突触电位。底部迹线是由注入电流直接进入RZ细胞之一诱发。注意增加突触电位的振幅和56小时培养后，改变动作电位波形。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig6highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig7.jpg" width="600px" /> 图7。水蛭神经节-体壁准备映射感受野（A）一种方法是伴随着1-3节清除体内壁的整个部分。在这里，在一边的根有BEEN切割和神经节已经被固定，以一侧。（B）固定，神经节在更高的放大倍率（C）另一种方法是在体壁和记录的完整神经节创建一个小的窗口，同时映射一个感受野（DG）各感觉神经元表现出对机械刺激的特征响应。（D）光触摸唤起了T细胞的活性，但不能在P或N个单元（E）的T细胞应答响应更强关闭刺激，活化在P细胞让路。由于刺激变得更加牢固的N个单元变为激活（FG） <a href="https://www.jove.com/files/ftp_upload/50631/50631fig7highres.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig8.jpg" width="600px" /> FIGURE 8。 诱发活性的水蛭神经节。连接词的细胞外刺激可以用来驱动突触传递的神经线。（A）这是通过附加抽吸电极的连接词之一，并施加一个小的电压进行。锋利的玻璃电极（箭头）可以看出，在刺穿神经节一Retzius细胞（B）电压-时间的痕迹，显示刺激伪迹后突触电位（黑色）和动作电位（红色）。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig8highres.jpg" target="_blank">点击这里查看大图像</a> 。 <img alt="图9" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig9.jpg" width="600px" /> 图9。染料注入到水蛭的神经元。（一）荧光黄可以通过使电流救援人员到场注入苏摩唉记录微电极。在注射后的水银灯泡和FITC过滤器设置可以被用于可视化荧光（B）染料已扩散到RZ轴突约30分钟。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig9highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis at JoVE.com

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

本文介绍了三种神经系统准备采用水蛭：细胞内记录从腹侧神经节的神经元，从腹侧神经节神经元培养，并从神经支配的皮肤感觉映射字段的补丁记录。

细胞内记录，感官字段映射，并在水蛭养殖鉴定神经元，医用水蛭</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

intracellular-recording-sensory-field-mapping-culturing-identified

Research

JoVE Journal

Neuroscience

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

19.6K 次观看.  University of Kentucky. 本文介绍了三种神经系统准备采用水蛭：细胞内记录从腹侧神经节的神经元，从腹侧神经节神经元培养，并从神经支配的皮肤感觉映射字段的补丁记录。 

视频: Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

电极的制作及植入海兔californica完整的，自由的行为的动物多通道的神经和肌肉的录音

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

科研

神经科学

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

由运动神经元调制生理，形态学和神经化学的表征

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

本地化的RNAi和药用水蛭异位基因表达

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

生物学

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

NRCC的膜片钳芯片上细胞的培养和电

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

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 ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

细胞内记录，感官字段映射，并在水蛭养殖鉴定神经元，医用水蛭

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此视频中的章节

电极的制作及植入海兔californica完整的，自由的行为的动物多通道的神经和肌肉的录音

由运动神经元调制生理，形态学和神经化学的表征

本地化的RNAi和药用水蛭异位基因表达

NRCC的膜片钳芯片上细胞的培养和电

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元

多单位记录方法来描述蝗虫的神经活动（

隔离的感觉神经元中

在两个神经元处理阶段在做人蜜蜂同时长期录音

斑马鱼

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议