我们感谢Hyewon库珀的插图。

1。解剖在本协议中使用的蟑螂生理盐水溶液具有下列组成： 蟑螂生理盐水溶液36：（克为100ml）中 210 mM氯化钠（1.227克） 2.9毫米氯化钾（0.0216克） 1.8毫米氯化钙2（0.0265克） 0.2毫米的NaH 2 PO 4•2H 2 O（0.0032克） 1.8毫米的Na 2 HPO 4•7H 2 O（0.0483克） （pH值7.2。调节pH，用1M的NaOH或1M的HCl）。 <ol><li>选择雄性蟑螂从贮水箱，具有强大的尾叶（ 图1）。相比于女性的男性的最后段狭窄，且不含卵巢和卵子的质量，男性更容易剖析。 P的男性美洲有一对尾叶之间的短测针。这些测针未在女性观察。 </li><li>切断翅膀，腿和头脚的身体，腹面朝上，以二SH内衬有机硅弹性体。 </li><li>用钳子拿起腹板和他们剪除用细剪刀，开始在后端和工作向前。总是用生理盐水保持内脏湿润，同时试图保持尾叶干。人们可以使用蜡或橡胶件定位在腹部向上，以防止盐水浸湿的尾叶。如果他们不被淋湿，干他们用一张纸巾。推到一边内脏和白质（脂肪体）。 VNC的是在该领域的中心，运行腹部的长度，并且应该有光泽的气管之间可见。神经索是半透明的，并且最初可能很难看到，直到照明的适当调整（ 图2）。不处理VNC用镊子或昆虫针，而是用玻璃探头操纵它。 </li><li>清除动物的气管系统尽可能最佳的用钳子神经线和一对精美的玻璃制品需要LES，非常仔细地分裂VNC连接词A6和A5或A5和A4神经节（ 图3）纵向之间。底座上的尾叶和腹部向上出盐水浴缩短昆虫标签和蜡或有机硅弹性体，可以以适合的制剂（ 图4A和B）的一个楔形的。要格外小心，在过去的腹节不损坏该项目进入神经 ​​节的神经塞卡尔（ 图2D和5）。 </li></ol> 2。细胞外记录<ol><li>解剖的制备，显微镜，和记录装置应该设置一个法拉第笼内，以阻止外部，特别是交流电源，电场可以重写信号从神经元（ 图6）。 </li><li>放置显微镜，使其俯瞰显微镜载物台。一旦该制剂放置在台上，调节高强度照明光束的位置对于它的最佳视觉效果。 </li><li>交流/直流差动放大器连接到集成数据记录单元（具体硬件和软件设置的详细信息之前已经描述了25）。头阶段保持抽吸电极应连接到放大器。银地线已涂有Cl -的插入更稳定的记录腹部的结果。其原因是，如果在体腔中的溶液不与在盘的浴液接触，以保持接地的记录电极相关联的流体。 </li><li>录制频率设置为4千赫。设定电压量程（y轴）为500 mV（这可以被调整以优化轨迹的可视化）。运行该录音软件在连续或示波器模式，以响应刺激记录神经活动。 </li><li>切VNC连接词接近A5的一个，然后将切口端连接到A6成吸电极。请务必为pULL生理盐水吸吸电极在神经前盖里面的银线。 </li><li>上到位于每个尾须毛干吸管吹空气。看是否刺激对尾须同侧的毛发所记录的连通性提供了比健侧一个不同的反应。采取的响应的幅度和峰值在刺激过程中给定时间间隔的数量的注释。 </li><li>将吸电极的塞卡尔神经进行录制。为了获得更好的贴合，​​开关电极尖端与一个较小的开口，如果必要的。 </li><li>切塞卡尔神经接近A6，然后吸了通向尾须神经。应该有动作电位的自发放电。现在，吹气到尾须，并注意反应。 </li></ol> 3。电刺激感觉神经，以确定招聘<ol><li>改变录音软件扫模式，这样它记录的痕迹（100-500 MSE角），每次刺激触发。 </li><li>刺激电极连接，刺激器的输出。 </li><li>与两个小钩引线或片段连接的刺激电缆。 </li><li>从激励器输出的BNC触发器连接到所述记录装置上的触发输入。 </li><li>下面的刺激参数应该唤起反应：持续时间：0.3秒;延迟：10毫秒;频率：1赫兹，电压：根据需要在录音得到的信号调节（刚刚超过阈值，并能够获得的最大响应）。我们没有理由去电压高于最大阈值高得多招聘作为一个高电压可损坏的神经。 </li><li>切塞卡尔神经的末梢尽可能使长期神经根可拉成的刺激吸引电极（ 图7，箭头）。可用于A6和A5或另一段更前壁之间的连通。 </li><li>设置录音吸电极，因此您可以PULL了切连通到电极。一定要拉一些生理盐水吸吸电极在神经前盖里面的银线。确保刺激电极也接地在洗澡液（在靠近A3腹部是理想的）。 </li><li>提供了一系列单一的刺激增加电压，直到动作电位出现在屏幕上。每个人都应该做的最小激励电压和持续时间的记录招募的响应。增加强度，直到连接词一个突触反应观察。从巨轴突的大穗（外接入点）先出现，然后其他较小的AP的也可以观察到。 </li></ol>

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作者宣称，有没有利益冲突。

其中一个原因，参展的技术为这个经典的准备是，尾叶系统一直并且仍然是研究解决神经电路的发展问题以及关于突触的修复和再生26-31题的活跃领域。唤起活性的蟑螂腹神经索的任一方法可以用来检验的药理剂或杀虫剂对神经系统功能的影响。这些实验是通过简单地溶解神经活性化学物质进入盐水进行。交换这种解决方案与正常洗澡介质后，改变诱发或自发的活动可能会同时从连接词或运动神经记录给对中枢神经系统功能的化学品的效果一致的读数进行观察。 正如在所有的神经生理学实验的一个常见问题是电气噪声。可能是在为这些准备信号质量的最大因素我S上的神经组织抽吸电极密封。一个密封不完全在塞卡尔神经或结缔组织画是理想的。记录，也可以与神经索和绝缘的VNC与矿物油和凡士林的混合物下放置双钩电极制成。该混合物可以被加载到一个注射器和神经索32周围排出。还仔细解剖这里是至关重要的，因为在中枢神经系统的任​​何准备。有些人可能会发现更容易通过解剖背角质层进入中枢神经系统。虽然这减小了损坏腹神经索的可能性也可以是更难以用这种方法除去所有的内脏。 这里不描述，但该制剂是适合于细胞内记录的巨型的interneurons 32,33。整个神经索，也可以拆下来同时容纳几个记录和刺激电极。在触角叶，蘑菇博其实探索DY，和其他中枢神经系统前部结构仍在进行中34-35。而蟑螂中枢神经系统继续阐明现代神经生物学研究光这个特殊的准备是很简单的本科学术实验室使用。

有超过4000种的蟑螂，但只有约30是家喻户晓的害虫。也许最认可的是名不副实的美洲大蠊美洲大蠊起源于非洲，现在发现几乎无处不在这个星球上。除了 ​​它的运行速度快1，回避行为，在热带P。美洲是能够飞行2,3。 蟑螂的中枢神经系统（CNS）的主要特点是它的分段控制的性质和权力下放进程4,5。脑，胸，腹部神经节是由成对的interganglionic连接词连接在一起形成腹神经索（VNC）。 在每个段的神经节整合中心。它们是由含有负责血 - 脑屏障渗透性只是它们下面的细胞外，皮层区域，以及神经元原稿的胞体的内廷中的神经节。这些胞体可能属于的interneurons，调节神经元，或运动神经元。他们提供保持原点（本地interneuron）的神经节内的神经轴突，或中枢神经系统（interganglionic的interneurons）的神经节或周围肌肉细胞（运动神经元）的终止之间的轴突投射。大多数胞体位于腹侧或ventrolaterally在皮层神经节5。配对，interganglionic连接词只包含轴突和无神经元胞体。 一个神经节的神经纤维含有胶质细胞（神经胶质），轴突束，轴突束和神经元的树突（突起）。该神经纤维缺乏是神经元细胞体。这就是神经细胞和整合投入之间的直接突触的沟通出现神经节内的区域。 美洲大蠊P的能力美洲检测和突然接近的捕食者回应（脚，汉d， 等等 。）已被归因于一种条件反射电路，包括尾叶和巨纤维系统6,7。的尾叶是一对位于上腹部的端部（ 图1）喇叭状，风敏感结构。在体育美洲每个尾须的腹面含有约200毫（线程），它们分为14列毛。这些列的九个可以在不同的动物被一致认定根据相关联的受体细胞和轴突的响应特性。每个头发是在一个插座，它允许弯曲最容易在一个平面上，作为列特异性的。头发在沿其平面沿一个方向的运动导致在受体细胞去极化和脉冲串的动作电位（AP）的中的感觉神经元。移动在相反的方向抑制任何正在进行的自发的AP 8。偏转的响应和方向性的优选平面是不同在每列中。因此，filifor米毛发受体复合物不仅用于检测空气的运动，但也为“编码”，在接入点，从该气流源自的方向的形式负责任。此信息由中枢神经系统的处理结果在“合适的”逃避反应6,7。在感觉毛的这种功能，柱状特异性保存从动物到动物。 每个丝状毛发的受体细胞是负责转导毛发的机械偏转成神经事件（导致受影响的受体细胞的轴突9的突发或抑制该接入点通过塞卡尔行进到终端腹腔神经节（A6）神经XI，在那里它们与腹神经索（VNC）的巨轴突突触的巨轴突被认为是负责运动神经元的传输和随后的激励而导致的逃避行为6,10,11。 行为延迟Øf P的逃避反应美洲是最短的任何动物7中的一个。行为延时是一个刺激的在机械感受器的到来和逸出反应的开始之间的时间。在使用高速摄影记录从一个攻击蟾蜍的企图逃跑的实验中，蟑螂被观察到开始轮到它离蟾蜍在大约40毫秒（时间从舌头延伸的开始蟑螂运动7,12。使用控制喷风，行为延迟可以减少到11毫秒，其他的实验表明，12毫米/毫秒的最小风速度粉扑（用600毫米/毫秒2的加速度）可以唤起一个转义响应，而更低的速度（0.1毫米/秒）引起缓缓地走蟑螂停止移动12。 强相关性是巨大的光纤系统和逃逸行为之间通常存在已经有据可查13,14。在研究所元代其中一个特定的细胞是必要的和足够的唤起特定行为的小区被称为命令神经元15,16。巨人的interneurons（GIS）在P的风遁电路美洲是没有必要的反射。已经烧蚀实验动物大兵仍然表现，因此这些美国大兵不考虑命令的神经元17,18的逃避行为。切断颈连接词是喙的感觉电路也会影响行为，这表明从大脑降序输入有对逃逸19的方向的效果。精细控制和冗余的这些方面是最重要的有机体的生存，并通过生物 ​​胺20的补充，神经化学调制。 该P.美洲神经索的准备一直是neuroethologists一个优雅的模型系统在过去的几十年开始，罗德&lt;的开创性工作燮&gt; 21。它允许学生记录，显示和分析由巨型的interneurons初级感觉活动，所得响应他们的输入22,23,24。除了传达的想法，可识别的神经回路背后的行为反应的环境，这些练习应该灌输给这个普通的家庭害虫作出的生物贡献的赞赏。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>Dow Corning</td>
			<td>182 silicone kit</td>
			<td>182 silicone elastomer kit</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>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>71505</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#8226;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S9390</td>
			<td></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></td>
			<td></td>
			<td></td>
			<td>Material Name</td>
		</tr>
		<tr>
			<td>Dissecting tools</td>
			<td>World Precision Instruments</td>
			<td>assortment</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</td>
			<td>World Precision Instruments</td>
			<td>PZMIII-BS</td>
			<td></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>Can fix for base or on a metal rod</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>AD Instruments</td>
			<td>27T</td>
			<td></td>
		</tr>
		<tr>
			<td>Head stage</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Comes with AC/DC amplifier</td>
		</tr>
		<tr>
			<td>LabChart7</td>
			<td>AD Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical leads</td>
			<td>any company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tools</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</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&#39;s brand)</td>
			<td>&#188; 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>

neural circuit recording from an intact cockroach nervous system

蟑螂腹神经索制剂是易于处理的系统，用于神经行为学实验，神经网络建模和测试杀虫剂的生理效应。本文介绍了可用于测定昆虫的神经系统如何响应环境扰动蟑螂感官方式的范围。这里强调的是由尾叶在美洲大蠊巨头光纤传输介导的逃避行为。这在原位制备只需要适度的解剖技能和专长电产生的神经元活动的可重复的记录。肽或其它化学试剂然后可以在与生理盐水溶液直接施加到神经系统。杀虫剂也可事先对解剖给药和避让电路可以作为中枢神经系统的兴奋性状态的代理。在这样的背景下在此描述的测定法也是有用的对r感兴趣的肢体再生和神经系统发育的演化esearchers为其P.美洲是一个既定的模式生物。

对尾叶毛发通过空气的吹刺激引起，可以使用附加或腹腔神经节或神经塞卡尔本身（ 如图8所示 ）之间的联结外抽吸电极进行记录的初级感觉神经元的放电。从两个区域记录尖峰振幅的几个微伏到毫伏的数量级。由于在神经节感觉整合在复合动作电位或从塞卡尔神经记录个人尖峰观察到尖峰的个数比在记录从连接词观察到显着更高。但是也可以注意到，基本上在记录由于在电极和神经组织之间的紧密密封噪声在结缔组织少。 通过喷着气在尾叶大穗可以在连接词（ 图8A）进行观察。使用该刺激方法，A3和A4之间的录音典型<html> LY显示巨头interneuron（S）的大穗的特性。从塞卡尔神经录制时擦身体用钳子的尾叶生产活动的一个强有力的胸围（ 图8B 1）。在另一个记录，2喷空气的每个产生于塞卡尔神经快速猛击响应（ 图8B 2）。当电刺激神经 ​​塞卡尔在A3和A4之间的连通的抽吸电极和记录，可以观察到一个阈值的刺激，以诱发反应（ 图8C 1）。该塞卡尔神经的电刺激诱 ​​发清楚在连接词的响应可被量化为与药剂或局部环境包围，如温度操控的研究（ 图8C 2）。 <html> fig1.jpg“/&gt; 图1。 美洲大蠊具有完好尾叶。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50584/50584fig2.jpg" /> 图2。蟑螂的神经索的观察与腹角质层去除腹面观（A）。由箭头所概述的段的放大图可见于（B）。 （C）中的连接词是A4和A3之间洒落用玻璃探头。 第六届腹神经节中显示（D）与两个塞卡尔离开神经在尾端。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/50584/50584fig3.jpg" /> 图3。蟑螂的神经索的示意图腹面观。 YS“&gt; <img alt="图4" src="/files/ftp_upload/50584/50584fig4.jpg" /> 图4，尾叶是向上时出盐水浴的。打开的腹部可以用生理盐水（A）与蟑螂的尾端被提升用小楔形形片有机硅弹性体，以保持尾叶出被洪水淹没镀液的（B）。 <img alt="图5" src="/files/ftp_upload/50584/50584fig5.jpg" /> 图5， 第 6腹节与塞卡尔神经（箭头概述）。 <img alt="图6" fo:content-width="6in" src="/files/ftp_upload/50584/50584fig6.jpg" /> 图6。设备设置。 <a href="https://www.jove.com/files/ftp_upload/50584/50584fig6large.jpg" target="_blank">点击这里查看拉尔</a>GER的身影。 <img alt="图7" src="/files/ftp_upload/50584/50584fig7.jpg" /> 图7。刺激和记录电极设置。 <img alt="图8" fo:content-width="5.75in" src="/files/ftp_upload/50584/50584fig8.jpg" /> 图8的连接词和塞卡尔神经与各种刺激手续神经录音。录音与A3和A4之间的连接词吸电极，而膨化的空气中，尾叶（A）。从主塞卡尔神经元具有吸电极记录，而无论是身体上摩擦（B 1）或提供空气喷（B 2）的结果在活动中迅速爆发的塞卡尔神经。电刺激塞卡尔神经产生的连接词回应（ <strong&gt; c 1）。请注意，在刺激强度逐渐增加（箭头表示刺激伪迹的振幅）和下述诱发反应的强度。该塞卡尔神经的电刺激提供了一个相对更可控的刺激塞卡尔神经在刺激一致性量化的反应（C 2）的装置。

Watch this Scientific Journal Video about Neural Circuit Recording from an Intact Cockroach Nervous System at JoVE.com

Neural Circuit Recording from an Intact Cockroach Nervous System

本文介绍了蟑螂腹神经索解剖，并从塞卡尔神经和连接词外记录。由塞卡尔神经或尾叶的直接机械刺激的电刺激产生诱发反应。

神经电路记录从一个完整的蟑螂神经系统

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

neural-circuit-recording-from-an-intact-cockroach-nervous-system

Research

JoVE Journal

Neuroscience

32.1K 次观看.  University of Kentucky. 本文介绍了蟑螂腹神经索解剖，并从塞卡尔神经和连接词外记录。由塞卡尔神经或尾叶的直接机械刺激的电刺激产生诱发反应。 

视频: Neural Circuit Recording from an Intact Cockroach Nervous System

Revealing Neural Circuit Topography in Multi-Color

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.

We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.

多色揭示神经回路地形

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

科研

神经科学

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

记录神经反应和注射示踪剂一个清醒小鼠的制备

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

鉴定转基因鱼的完整的大脑中的神经元的电活动记录

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三星果蝇中枢神经系统活动的电生理记录

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

使用电压敏感染料在脑切片中进行宽场单光子光记录

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.