我们要承认莱纳斯D.太阳和Jeenah荣，起源于使用电脑设计，3 - D印在我们的实验室中的驱动器，记录小鼠。此外，我们要感谢所有过去和现在的威尔逊实验室成员的贡献微驱动器和四极管在实验室的技术开发。

<ol><li> 微驱动器的阵列概述 一个完整的微型驱动器阵列由几个主要组成部分（图1）: <ol><li>一个拥有21独立可移动的微型驱动器的计算机设计的塑料底座。 </li><li>在收集了一套固定的四极导管的基底部的套管。 </li><li> 21微驱动器，各携带一个四极管和可以单独驱动使用一个螺丝钉。 </li><li>电极为四极管和前置放大器之间的互连接口板。 </li><li>保护锥（不会显示在图1） </li><li>在完全组装的微型驱动器阵列，塑料基地，拥有所有的微型驱动器。每个微驱动器进行单一的四极。四极是电气连接电极接口板的微型驱动器阵列的顶部，并通过固定导管，退出对​​大脑的微驱动器阵列的底部。打开每个微驱动器的进步或缩回的四极螺钉。 </li></ol></li> <li> 塑料底座的设计 在一个3 - D CAD软件包（SolidWorks的），我们设计的微型驱动器组装基地。这房子的设计过程的优点是快速周转和设计的灵活性。在威尔逊实验室，我们设计了许多变种这一部分，对每个实验的具体要求，面向。 3 - D模型文件发送到使用光固化树脂激光固化的液体，它是从印刷服务局。周转时间只有2天。由此产生的部分是轻量级的，可重复使用的的，可以加工成一个线程持有。在这个协议中，我们将使用一个驱动器是一个单一的录制现场，并拥有21个微型驱动器设计的基础。 SolidWorks设计文件要求提供。 </li> <li> 塑料底座的制备 塑料基处理，以适应微驱动器和螺丝连接电极接口板和防护锥。是不可能可靠地打印在塑料基通孔顺利，所以我们设计不足的孔，然后展开钻孔。小心地将现有的孔与轴前的钻头钻在非常低的速度。一个变量的速度/转矩的手钻这个任务。 <ol><li>钻孔的微驱动器的螺丝，使用61位（外圈）的21个试点孔。 </li><li>微型驱动器使用65位的支撑管（内圈）钻21孔。 </li><li>钻的孔将用于连接电极的接口板和使用防护锥一个1.55毫米的位。 </li><li>按适合使用镊子或钳子孔内环每一个套管（5毫米长20嘎薄壁不锈钢管），因此，它是与塑料底座平齐。袖子确保平稳运动的微型驱动器支撑管。 接下来，所有的孔，将举行螺钉被挖掘出来。在印刷塑料，螺纹自成形水龙头比典型的线程切割水龙头更耐用的线程的结果。这种风格的自来水需要较大的导孔。使用手持针老虎钳按住水龙头: </li> <li>点选微型驱动器使用的自来水，与正在使用的螺丝大小（M1.2x0.25）和干式润滑剂的螺丝的孔。为了帮助协调与先导孔的水龙头，插入旁边的袖子长度23日嘎管。 </li><li>我们的塑料底座的设计功能，为每一个微驱动器的螺丝孔，以确保初始线程在线程的过程中损坏的自来水起动。毕竟有螺纹孔，这些挖掘初学者可以很容易折断使用刀片。 </li><li>塔将于电极接口板和使用1-72挖掘保护锥螺丝的孔。 </li></ol></li> <li> 电极导管插入 <ol> <li>为了使所有固定电极导管插管，持有，削减了13 GA不锈钢管1.5厘米长片，并与DREMEL砂轮打磨表面的附着力，以加强牙科丙烯酸。 </li><li>在塑料底座的底部孔插入导管，确认它是与垂直轴驱动器的共线性，安全与牙科丙烯酸酯或环氧树脂。保留的暴露，以方便手术植入的导管5至7毫米。 </li><li>剪下21固定电极导管（灵活的聚酰亚胺管，内径:0.0071:0.0116，外径"）用刀片到6厘米的长度。 </li><li>饲料导管一次到驱动器基地，通过集热器底部的套管顶部的袖子。如有必要，扭结或过度弯曲和更换管目视检查。为了便于和四极导管插入平行排列，就可以开始与第一个整捆的21个导管送入从下面的集热器套管。然后，一次一个，从捆绑中删除一个导管和饲料通过孔塑料底座的顶部，它留在后面的捆绑。 </li><li>导管推下来，直到他们把塑料底座的顶部只有1-2毫米。应用少量的氰基丙烯酸酯胶薄管，因为他们退出的集热器套管，小心不要让任何胶水流进入导管年底。 </li><li>使用一个新的刀片切断导管束在集热器套管底部多余的。 </li> </ol></li><li> 微驱动器的制作 每个微驱动器包括一个定制加工的螺钉和空心支撑管（图2）。螺杆和支撑管连接在一起，嘴唇上的螺丝钉嵌入在丙烯酸，离开自由旋转螺丝（图2B）。螺纹螺杆塑料底座，然后带动了支撑管或向下转动螺丝时，提前或收回它携带的四极。为了便于调整行为动物，螺丝头是在一个半圆柱体的形状。制作自定义为这个螺丝起子: <ol><li> 15 GA不锈钢管切5厘米。 </li><li>以自定义的螺丝和研磨凸出唇。 </li><li>插入油管的螺丝，直到顶部的螺丝与管端平齐。 </li><li>粉碎周围螺纹管反复用一双​​钳使螺丝不能旋转套管。 </li><li>使用PIN老虎钳来保存自定义的螺丝刀。 编造的微驱动器，我们使用一个3 - D印刷的塑料模具与小水井（3毫米x 5毫米，2毫米深）。每口井有两个孔，在底部的表面，支撑管和螺丝，间距为2毫米。此外，模具可如聚甲醛工程塑料制造，使用一台钻床，创造良好，孔。 </li> <li>准备结算的支持套管使用了23 Ga的针和塑料底座，上面所述的螺丝孔，攻丝孔的模具。 </li><li>线的聚四氟乙烯润滑剂或凡士林薄膜，以便释放牙科丙烯酸固化后从模具以及​​内部.. </li><li>聚四氟乙烯润滑剂应用到一个螺丝钉，把它插入到模具使用定制螺丝刀，直到线程略低于井底。 </li><li>粗糙，用砂轮增强粘附牙科丙烯酸一个14毫米长的支撑管顶部2-3毫米（23 GA不锈钢管）。插入模具，直到导管顶部中间以及顶部和顶部的螺丝。 </li><li>倒入以及牙科丙烯酸。是用来搅拌的议案，以确保牙科丙烯酸到周围所有的螺丝和支撑管的空间流动。删除任何气泡，用小针。 </li><li>等待，直到彻底治愈牙科丙烯酸（15-30分钟），然后从模具中删除反顺时针转动螺丝微型驱动器。 </li><li>重要的是做一个微驱动器的质量检查。检查治愈裂缝和/或气泡，这通常是由牙科丙烯酸，过厚或过薄造成牙齿丙烯酸。另外，确保螺丝和支撑管是相互平行的，螺丝轮流顺利。从牙科丙烯酸及循环再造的，不符合这些标准的微型驱动器的螺丝和支撑管可以被破解。 </li><li>重复步骤5.6到5.11，直到你有21个微型驱动器。 </li></ol></li> <li> 微驱动器阵列的最后组装<ol> <li>下到每个微驱动器的塑料驱动器的基本。每个微驱动器支撑管内外套筒和超过其固定导管应平稳移动。检查，以确保导管不扣微驱动器正在降低。 </li><li>随着微型驱动器完全降低，加一层薄薄的牙科丙烯酸的驱动器的基本内部固定在电极导管。 </li><li>请注意，降低之前，所有的方式加入牙科丙烯酸的微型硬盘的失败将导致丙烯酸了袖子和微驱动器运动的阻碍排汗一路。 </li><li>标签的微驱动器1-21。 </li><li>打开驱动器阵列倒挂，采取导管束的图片。这张照片将用于地图位置对应到每个微驱动器的导管。 </li><li>所有的微型驱动器提高几个毫米。 </li><li>插入聚酰亚胺载体到每个从驱动器的基本底部的导管管（0.005"），让承运人管延长1-2毫米的全面降低了微驱动器和记录的顶部，从照片上的身份相应的微型驱动器。 </li><li>粘承运人管使用5分钟环氧树脂或氰基丙烯酸酯胶的微驱动器支撑管。注胶或环氧树脂，太薄会流下来了支持套管和妨碍自由运动的微型驱动器，或者更糟，解决它的引导导管。如果发生这种情况，特别是微型驱动单元不能使用。 </li><li>全面降低所有的微型驱动器。在集热器套管底部平齐使用新鲜的刀片切断所有的运营商管。 </li><li>最后，安装Neuralynx电极接口板驱动器的基本使用两个螺钉（大小:1-72，长度:3 / 16"）。 </li></ol></li></ol>在这一点上，准备与四极管装入驱动器阵列。制造的四极管，如何加载到微驱动器，以及如何准备手术植入驱动器阵列，随后视频文章的主题。 <img src="/files/ftp_upload/1094/Figure1.png" alt="图1" /> 图1。成品微型驱动器阵列的型号。一个微驱动器阵列由几个主要部分组成:塑料驱动器基地;从四极管进入大脑一个收藏家套管; 21微驱动器，每个驱动器的单四极管;电极接口板连接到前置放大器组成。 <img src="/files/ftp_upload/1094/Figure2.png" alt="图2" /> 图2定制微驱动器的螺丝。A.自定义的螺杆设计技术图纸。螺杆采用了螺纹部分（M1.2x0.25;直径1.2毫米，0.25毫米/转），一个半缸盖匹配定制螺丝刀和顺利与唇的一部分（见详细）B.模型完整的微驱动器。牙科水泥支撑管和定制螺丝连接。唇部上的螺丝钉是嵌入在牙科水泥和自由旋转允许驾驶时向上或向下的支撑管。 附加文件我们设计印刷的驱动器基地，保护锥和2008年在SolidWorks 3D CAD软件的微型驱动器建设的模具: <ul style="list-style-type:none;"><li>下载文件: <a href="/files/ftp_upload/1094/DriveBase.SLDPRT">DriveBase.sldprt</a>其中包含驱动器的基础和保护锥和第模型。 </li><li>下载文件: <a href="/files/ftp_upload/1094/MicroDriveMold.SLDPRT">MicroDriveMold.sldprt</a>其中包含的微驱动器的模具模型</li></ul>三维打印服务局不能直接处理原始的SolidWorks文件，而是需要在光固化CAD格式（STL）的设计文件。人谁没有访问SolidWorks软件，但仍然想用视频文章中使用的印刷部分，我们包括在STL格式以及设计: <ul style="list-style-type:none;"><li>下载文件: <a href="/files/ftp_upload/1094/DriveBase.STL">DriveBase.stl</a>其中包含驱动器的基本模型</li><li>其中包含的微型驱动器模具模式，下载文件<a href="/files/ftp_upload/1094/MicroDriveMold.STL">:</a> MicroDriveMold.stl </li><li>下载文件: <a href="/files/ftp_upload/1094/ProtectionCap.STL">ProtectionCap.stl</a>其中包含第防护锥模型</li><li>下载文件: <a href="/files/ftp_upload/1094/ProtectionCone.STL">ProtectionCone.stl</a>其中包含保护锥模型</li></ul>

<ol>
	<li>Yamamoto, J., Wilson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+chronically+implantable+precision+motorized+microdrive+array+for+freely+behaving+animals.">Large-scale chronically implantable precision motorized microdrive array for freely behaving animals.</a> J. Neurophysiol. 100 (4), 2430-2440 (2008).</li></ol>

本协议描述了微驱动器的建设过程中的一般特点。它已成功地修改，以产生多个录音的目标或在小鼠体内的录音较小的阵列驱动器阵列。除了海马记录，许多研究人员使用这些驱动器的目标皮层和皮层结构。可能需要使用较长的螺丝，以记录从更深层次的大脑结构。随着大量修改的驱动器的基本，机动驱动器可用于代替手工打开的，可以更精确，远程四极调整（山本和威尔逊，2008年）。定制固定...

<table>
<tbody>
	<tr>
<td>3D-printed plastic parts</td>
<td>Material</td>
<td>American Precision Prototyping (www.approto.com)</td>
<td>&nbsp;</td>
<td>Parts are printed by stereolithography (SLA) using DSM 14120 'ABS-like', DSM 11120 'Watershed' or Accura 55 resins. Request high resolution, no sandblasting.</td>
</tr>
<tr>
<td>stainless steel tubing</td>
<td>Material</td>
<td>Small Parts (www.smallparts.com)</td>
<td>&nbsp;</td>
<td>Cut to custom length and deburred. Sizes: 13 Ga (1pc @ 10 mm), 15 Ga (1pc @ 50 mm), 20 Ga thin wall (21pcs @ 5 mm), 23 Ga (21pcs @ 14 mm) </td>
</tr>
<tr>
<td>Dental cement powder</td>
<td>Material</td>
<td>A-M Systems (www.a-msystems.com)</td>
<td>525000</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dental cement solvent</td>
<td>Material</td>
<td>A-M Systems</td>
<td>526000</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Electrode interface board</td>
<td>Material</td>
<td>Neuralynx (www.neuralynx.com)</td>
<td>EIB-81</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>micro-drive screws</td>
<td>Material</td>
<td>Advanced Machining and Tooling (www.amtmfg.com)</td>
<td>&nbsp;</td>
<td>Custom made screws with M1.2x0.25 threads (1.2mm major diameter; 0.25 mm thread pitch) </td>
</tr>
<tr>
<td>'Tap-Ease' tapping lubricant </td>
<td>Tool</td>
<td>AGS Company</td>
<td>TA-2</td>
<td>Available from MSC Industrial Supply (www.mscdirect.com)</td>
</tr>
<tr>
<td>Polyimide tubing </td>
<td>Material</td>
<td>IWG High Performance Conductors (www.iwghpc.com)</td>
<td>&nbsp;</td>
<td>Sizes:
30 Ga (outer diam: 0.0071", inner diam: 0.0116") 
5 mil (nominal outer diam: 0.0055", wall: 0.0010") </td>
</tr>
<tr>
<td>Cordless drill/driver </td>
<td>Tool</td>
<td>DeWalt (www.dewalt.com)</td>
<td>DC750KA </td>
<td>Any drill/driver with low-speed setting will do.</td>
</tr>
<tr>
<td>Variable-Speed Rotary Tool Kit with Flex-Shaft </td>
<td>Tool</td>
<td>Dremel (www.dremel.com)</td>
<td>3956-02</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Razor blades </td>
<td>Tool</td>
<td>VWR (www.vwr.com)</td>
<td>55411-050</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Watch glass </td>
<td>Tool</td>
<td>VWR</td>
<td>66112-107</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tap for custom screws</td>
<td>Tool</td>
<td>Balax</td>
<td>01302</td>
<td>M1.2x0.25 Thredfloer thread-forming tap. Available from MSC Industrial Supply.</td>
</tr>
<tr>
<td>Taps</td>
<td>Tool</td>
<td>Small Parts</td>
<td>HSBT-0172, HSBT-0080 </td>
<td>1-72, 0-80 bottoming taps</td>
</tr>
<tr>
<td>Pin vise</td>
<td>Tool</td>
<td>Vargus</td>
<td>3SHC1--I </td>
<td>To hold taps. Available from MSC Industrial Supply</td>
</tr>
<tr>
<td>Drill bits</td>
<td>Tool</td>
<td>Small Parts</td>
<td>&nbsp;</td>
<td>Sizes: 65, 61, 1.55mm</td>
</tr>
<tr>
<td>Benchtop vise</td>
<td>Tool</td>
<td>Panavise</td>
<td>301</td>
<td>Available from DigiKey (www.digikey.com).</td>
</tr>
<tr>
<td>No.2 Dumont forceps</td>
<td>Tool</td>
<td>Stoelting (www.stoeltingco.com)</td>
<td>52100-27</td>
<td>Used for inserting 20 Ga thin wall sleeves.</td>
</tr>
<tr>
<td>5-minute epoxy</td>
<td>Tool</td>
<td>Allied Electronics (www.alliedelec.com)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Accu-Tek Carbofib Tip Tweezers</td>
<td>Tool</td>
<td>Aven (www.aveninc.com)</td>
<td>18768 (Pattern 304)</td>
<td>Used for handling polyimide tubing.</td>
</tr>
<tr>
<td>Micro Dissecting Scissors</td>
<td>Tool</td>
<td>Biomedical Research Instruments (www.biomedinstr.com)</td>
<td>25-1000</td>
<td>Used for cutting polyimide carrier tubes</td>
</tr>
<tr>
<td>Xcelite 378M Pliers</td>
<td>Tool</td>
<td>Newark</td>
<td>96F8903</td>
<td>Used for handling stainless steel tubing and for construction of custom screwdriver.</td>
</tr>
<tr>
<td>mini pin vise</td>
<td>Tool</td>
<td>MSC Industrial Supply (www.mscdirect.com)</td>
<td>00920314 </td>
<td>Used as a handle for custom made screwdriver.</td>
</tr>		
</tbody>
</table>

micro-drive array for chronic in vivo recording: drive fabrication

人口众多的神经元的慢性记录是一个有价值的技术研究在清醒行为大鼠的神经回路的功能。轻量级的录音设备，携带了四极管的高密度阵列允许为几十到数百个单个神经元活动的同步监测。在这里，我们描述了一个与二十一独立可移动的微型驱动器的微型驱动器阵列的制造协议。使用该设备已成功地从海马和皮层神经元在我们的实验室记录。我们将展示如何编写自定义设计，三维塑料印刷基地，将举行的微驱动器。我们将演示如何构建个人的微型驱动器和如何组装完整的微型驱动器阵列。进一步的准备手术植入的驱动器阵列，如四极管制造的四极管装入驱动器阵列和镀金，是覆盖在随后的视频文章。

Watch this Scientific Journal Video about Micro-drive Array for Chronic in vivo Recording: Drive Fabrication at JoVE.com

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

在这个协议中，我们将演示如何制造一个微驱动器阵列，在大鼠慢性电生理记录。

慢性的微型驱动器阵列在体内录音:驱动器的制造

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

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Biology

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

25.2K 次观看.  MIT - Massachusetts Institute of Technology. 在这个协议中，我们将演示如何制造一个微驱动器阵列，在大鼠慢性电生理记录。

视频: Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Population Replacement Strategies for Controlling Vector Populations and the Use of Wolbachia pipientis for Genetic Drive

In this video, Jason Rasgon discusses population replacement strategies to control vector-borne diseases such as malaria and dengue. "Population replacement" is the replacement of wild vector populations (that are competent to transmit pathogens) with those that are not competent to transmit pathogens. There are several theoretical strategies to accomplish this. One is to exploit the maternally-inherited symbiotic bacteria Wolbachia pipientis. Wolbachia is a widespread reproductive parasite that spreads in a selfish manner at the extent of its host's fitness. Jason Rasgon discusses, in detail, the basic biology of this bacterial symbiont and various ways to use it for control of vector-borne diseases.

In this interview, Jason Rasgon explains the concept of genetic drive and the characteristics of an effective gene drive system. The use of the endosymbiotic bacterium, Wolbachia pipientis, as a means to spread genes through mosquito populations, is hypothesized.

人口控制媒介种群和遗传的驱动器使用的Wolbachia pipientis替换策略

In this video, Jason Rasgon discusses population replacement strategies to control vector-borne diseases such as malaria and dengue. "Population ...

科研

生物学

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

光固化牙科粘合树脂安装电极或微透析探针在慢性实验中的应用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

一种慢性脑功能成像开颅手术过程

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential amplitude of action potential signatures over the channels of a tetrode allows for the isolation of single-unit activity from multi-unit signals.  The ability to precisely control the stereotaxic location and depth of the tetrode is critical for studying coordinated neural activity across brain regions.  In combination with a micro-drive array, it is possible to achieve precise placement and stable control of many tetrodes over the course of days to weeks.  In this protocol, we demonstrate how to fabricate and condition tetrodes using basic tools and materials, install the tetrodes into a multi-drive tetrode array for chronic in-vivo recording in the rat, make ground wire connections to the micro-drive array, and attach a protective cone onto the micro-drive array in order to protect the tetrodes from physical contact with the environment.



Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

慢性的微型驱动器阵列在体内录音:四极大会

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

在体内的器官型小鼠视网膜Wholemount文化

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

在小鼠体内的动态心电图记录

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

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 ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

阵列比较基因组杂交（CGH阵列），用于检测基因拷贝数变异的

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

测量DNA损伤与修复小鼠脾细胞慢性后<em&gt;在体内</em&gt;暴露于β和γ辐射剂量很低

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...