这项工作是由国立卫生研究院授予EY005102 SCM和EY018640中华人民共和国

1。完整的金鱼，老鼠和兔子的神经视网膜的准备<ol><li>在恒定的环境（背景）照明下执行包括以下步骤中所描述的，“2）剪切加载”。请注意，本协议的目的，程序为执行在不断的黑暗（即≤0.0001勒克斯）非常暗（即“暗”）的条件下使用夜视红外护目镜，但其他恒定的环境光照强度较高水平可以用来代替来确定的其他环境照明水平上的差距交界示踪耦合的影响。 </li><li>暗适应手术前至少1小时的实验动物（金鱼，老鼠或兔子）。 </li><li>正如先前所述7，深受麻醉金鱼放置在tricaine磺酸（MS222; 150毫克/大号缓冲（碳酸氢钠含）的鱼缸水），并深深地麻醉小鼠氯胺酮（100毫克/公斤，IP）和rompun（10毫克/千克，IP）。深麻醉与聚氨酯兔（负荷剂量：2.0克/公斤，IP），还可以使用本地眶内麻醉（2％Xylocaine），描述以前8。所有涉及护理和使用动物实验程序应按照联邦指导方针和本地大学动物护理和使用委员会进行审查和批准。 （注：在这里描述的实验，所有涉及鱼的关心和使用的实验程序，小鼠和家兔，按照NIH的指引，并进行了审查和美国俄亥俄州立大学的体制动物护理和使用委员会批准。） </li><li>对于鱼，小鼠和家兔，眼球摘除后，删除每个眼球前部。然后，放置了一块滤纸上的眼睛后部的顶端和反转滤纸和眼睛，使滤纸，眼睛的后部是下方。然后，用细钳解剖从眼睛的后部轻轻地剥离了色素上皮/脉络膜/巩膜，视网膜的神经，这是附着在滤纸，这是连接到对方的完整的神经视网膜。这样做是用细弹簧加载剪刀，剪断视神经。视网膜神经，面向感光面朝上，现在应该可以连接到滤纸和眼睛休息分开。 </li></ol> 2。切负荷<ol><li>淹没的视网膜在一个6孔钢板在恒定的环境（背景）照明（例如，在很暗（即“暗”）的条件）与下30分钟（〜5毫升/以及）或无含氧林格的解决方案（见表1）测试药物。保持密封的用封口膜6孔钢板鱼视网膜中含氧（5％的CO 2 / 95％O 2）碳酸氢钠，基于林格的22 °彗星，并维持在36℃在一个5％的CO小鼠和兔视网膜 2 / 95％的O <suB&gt; 2的孵化器。当测试使用药物，它应该是目前在所有的后续步骤，直到固定</li><li>溶解neurobiotin（0.5％），生物素化的分子，在林格氏液，立即通过切割视网膜前的准备示踪剂溶液（通常为100μL，）。请注意，0.5％罗丹明葡聚糖（高（&gt; 10,000）兆瓦）可添加到解决方案。由于其分子量高，罗丹明葡聚糖不跨缝隙连接和唯一的标签切已受损的细胞。 如图。 3，这是一个有用的的方法来区分已经积累了neurobiotin，因为他们已经从那些已经积累了通过缝隙连接扩散的示踪剂，受伤细胞。 </li><li>放在玻璃（或塑料）培养皿neurobiotin解决方案，挖掘在纸巾上的滤纸上，视网膜连接，去除多余的林格氏液，浸在neurobiotin解决方案刀片（或其他锋利的刀刃）在n请通过放射状切开视网膜和滤纸。如果首选，垫片可用于削减通过视网膜，但不是滤纸。切口应面向垂直视网膜表面。浸在neurobiotin解决方案刀片再次削减视网膜第二次。重复此高达4％削减（ 见图1）视网膜。使用解剖显微镜时，通过鼠标和其他小的视网膜剃须刀削减。 </li><li> 如图。 1，再次淹没在新鲜林格液的视网膜，或没有试药，用6板（5毫升林格氏液/孔） 。视网膜孵育15分钟，以便加载和扩散。 </li><li> 5分钟，每个试药带或不带新鲜林格液洗三次。 </li><li> 0.1 M磷酸缓冲液（PBS，pH值7.4）的4％多聚甲醛在室温下1小时修复视网膜。 </li><li>用0.1 M PBS清洗一夜之间4 ° C。 </li><li>翌日，反应瓦特第i个C. 2％链霉亲和Alexa的488（终浓度为10微克/毫升）在0.1M PBS + 0.3％Triton X - 100的的，并保持在4℃过夜</li><li> 0.1M PBS在室温下每10分钟洗三次</li><li> PBS中，视网膜脱离滤纸，然后，用细刷，放置在视网膜上，面向感光面朝上，到一个幻灯片。 </li><li>轻轻Vectashield安装媒介（媒介实验室，伯林格姆，CA）的安装。 </li><li>激光扫描共聚焦显微镜（例如蔡司510元），在每一个实验条件相同的放大倍率，分辨率和设置的图片，让你可以比较不同的实验条件（如测试药物;光照条件）的影响的程度缝隙连接示踪剂耦合（ 图2A - C和图4a - C的 ）。虽然单个图像可能包含所有的标记细胞，它是建议您收集感兴趣的领域的Z - Stack与它折叠成一个单一的形象。 </li></ol> 3。定量示踪耦合使用ImageJ <ol><li>在LSM的图像浏览器，打开图像，单击“导出”，并保存为未压缩文​​件为TIF - 16位图片。比例尺应包括在本TIF图像。 </li><li>使用NIH ImageJ软件，测量荧光强度从低倍率的整个安装视网膜图像Alexa的- 488 -标记neurobiotin。使用ImageJ软件打开TIF文件，然后对应的比例尺绘制一条直线。转到分析，设置规模和输入已知的距离和长度单位，然后单击确定。现在，测量结果可以在校准的单位，如毫米，。 </li><li>选择使用矩形选择工具的利益区域。荧光峰应定位在您选择的左边框。确保有没有合适的边境附近的荧光信号。如果没有，活动图像（影像，然后将其旋转）旋转。 </li><li>点击分析和剧情简介。你应该看到从左边到右边的指数衰减曲线。 </li><li>在弹出窗口中单击“复制并粘贴在Excel中。现在你看到两列显示的距离，从切（左栏）和荧光强度作为距离的函数从削减（右列）。 </li><li>最大荧光值除以每个原始的荧光值，以获得相对荧光强度。 </li><li>复制两列对应切（X）和相对荧光强度（Y）的距离，然后粘贴到Origin软件。 </li><li>配合指数衰减＃1的功能（图2D和图4D。），形式： Y = Y + Y 最大 * EXP（- X /λ） 其中Y是相对的荧光强度，Y o为背景荧光，Y max是最大的相对荧光，λ是SPACE（长）和X是从切的距离。 </li><li>比较不同实验条件下，采用t检验或方差分析（图2E和图 4E）的空间常数（λ）值。需要注意的是量化的替代技术已经由他人 13,14描述。 </li></ol> 4。代表性的成果感光细胞示踪耦合削减负荷确定的有代表性的例子是在图2和图 3 和图 4（兔）（鱼） 。使用相同的设置进行比较（ 图2A - C和图4A - C）和荧光强度的共聚焦图像绘制的距离，从削减的功能和装配号3.8上面显示的指数函数（ 图2D和图4D）。每个条件的空间内恒定值显示在图2E和4E，说明缝隙连接示踪剂耦合的程度是可以量化的使用削减加载技术。此外，高度重复性的结果。使用量化缝隙连接示踪剂耦合的程度的一种手段削减加载技术研究也发现，从削减在所有情况下，荧光强度随距离的函数指数验证7,8（见还图。 2D和4D），表明neurobiotin进入的光感受器，通过剃须刀切割和视网膜从其他网站不。此外，感光细胞示踪耦合的差异的定性类似的昼/夜观察金鱼示踪剂注射到单锥和截装载7证实，作为衡量感光耦合程度一个比较准确的方法削减负荷。 切- Loading技术也可以用来研究其他类型的视网膜电突触。例如， 图 5表明，兔A型（ 图5A）和B型（ 图5B），水平细胞表现出同源示踪耦合，以下切装和暗适应条件下的neurobiotin扩散。 <table border="1"><tbody><tr><td> 化合物 </td><td> 鱼 </td><td> 鼠标 </td><td> 兔 </td></tr><tr><td>氯化钠</td><td> 130 </td><td> 120 </td><td> 117 </td></tr><tr><td>碳酸氢钠3 </td><td> 20 </td><td> 25 </td><td> 30 </td></tr><tr><td>的NaH 2 PO 4 </td><td> - </td><td> 1 </td><td> 0.5 </td></tr><tr><td> KCL </td><td> 2.5 </td><td> 5 </td><td> 3.1 </td></tr><tr><td>血糖</td><td> 10 </td><td> 10 </td><td> 10 </td></tr><tr><td> MgCl 2的 </td><td> 1 </td><td> - </td><td> - </td></tr><tr><td>硫酸镁4 7H 2 O </td><td> - </td><td> 1 </td><td> 1.2 </td></tr><tr><td>谷氨酰胺</td><td> - </td><td> 0.1 </td><td> 0.1 </td></tr><tr><td> 氯化钙 </td><td> 0.7 </td><td> 2 </td><td> 2 </td></tr></tbody></table> 表1：林格氏液，金鱼，老鼠和兔视网膜的解决方案的组成。在MM的浓度。林格的解决方案是冒泡5％CO 2 / O 2的 95％，并保持在29 ° C（鱼）或36℃（哺乳动物） 。 “ - ”：不包括在这一物种的林格氏液。 <img alt="图1" src="/files/ftp_upload/3180/3180fig1.jpg" /> 图1：显示削减装载过程的流程图。经过隔离Øf的完整的神经视网膜，几个径向削减作了一个是第一次在0.5％的neurobiotin溶液蘸刀片。视网膜孵育示踪装载和扩散，然后固定在0.1M磷酸盐缓冲液（PB）的4％多聚甲醛（PFA）前洗净。示踪耦合研究使用链霉亲和共轭Alexa的- 488。 <img alt="图2" src="/files/ftp_upload/3180/3180fig2.jpg" /> 图2日夜在感光示踪耦合的区别是金鱼透露削减加载技术。感光细胞间隙连接neurobiotin示踪耦合是在夜间（二）在当天的广泛spiperone（10μM），选择性的多巴胺D 2受体拮抗剂（三）申请，但在当天没有在控制条件（一） 。四）规范化的相对荧光强度削减（在交流中的箭头表示）的距离的函数。 e）空间不断VALUES获得D和其他实验组（n = 4）的数据。 *** P &lt;0.001。 <img alt="图3" src="/files/ftp_upload/3180/3180fig3.jpg" /> 图3一个代表性的例子，显示在一个暗适应的金鱼视网膜的感光细胞在夜间加载切层以下neurobiotin和罗丹明葡聚糖溶液的荧光。罗丹明葡聚糖（红色显示），不漫过开放的间隙连接通道，由于其分子量高（&gt; 10,000 MW），只有标记细胞附近的切。 neurobiotin（以绿色显示）通过缝隙连接扩散，相比之下，可以看出，在感光细胞远离削减。切割的位置是在每个面板的箭头表示。比例尺：200微米。 <img alt="图4" src="/files/ftp_upload/3180/3180fig4.jpg" /> 图4。日夜不同ENCE兔视网膜感光示踪耦合透露削减加载技术。感光细胞间隙连接neurobiotin示踪耦合是在夜间（二）在当天的广泛spiperone（10μM）（三）申请，但在当天没有在控制条件（一）。在交流中，附近的切兔感光器的三维重建垂直意见。四）规范化的相对荧光强度作为削减的距离功能。五）获得D和其他实验组（n = 3）的数据空间内恒定值。 *** P &lt;0.001。 <img alt="图5" src="/files/ftp_upload/3180/3180fig5.jpg" /> 图5。剪切加载显示，暗适应兔横向细胞示踪加上。 A型（A）和B型（二）在暗适应兔视网膜水平细胞同源neurobiotin示踪耦合。

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+biotinylated+tracers+distinguishes+three+types+of+gap+junction+in+retina.">A series of biotinylated tracers distinguishes three types of gap junction in retina.</a> J. Neurosci. 20, 8629-8636 (2000).</li></ol>

没有利益冲突的声明。

这里描述的剪切加载方法是有用的和简单的技术来确定不同的光照条件下，在白天和黑夜的不同时期的视网膜神经细胞之间的缝隙连接示踪剂耦合的程度。这种技术的优点包括能够完整的视网膜组织中的神经元之间的缝隙连接示踪剂耦合程度量化各种光照条件下，白天和晚上的耦合神经元的小直径somata。在完整的组织分为两大类下降缝隙连接的研究技术的限制。首先，通过开放的缝隙连接示踪剂的扩散可能会比较难以观察到，由于A）小直径或与开放渠道和B），加上蜂窝车厢的相对体积1,14,15相关的费用。也就是说，从一个小细胞示踪扩散到一个较大的细胞或组耦合细胞，比赛ared从一个较大的细胞，以较小的细胞示踪扩散，可能会更加难以发现由于示踪稀释。第二，某些生理条件下，通过缝隙连接在完整组织的示踪扩散的程度可能无法准确反映的缝隙连接电导的实力相比，比较大的示踪通透性由于在小电流携带离子电导的差异，分子1,14,15。在一般情况下，示踪耦合的证据有力地表明运作，开放的间隙连接通道的存在，但某些生理条件下，示踪扩散可能不会发生或观察到，尽管电生理记录表明存在的运作，打开缺口路口。 这很可能削减加载技术还可以用于研究完整的组织从其他地区的中枢神经系统的神经元之间的缝隙连接示踪剂耦合程度TEM。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> Tricaine甲烷磺酸（MS222） </td><td> Sigma - Aldrich公司</td><td> A5040 </td><td> 150毫克/ L的缓冲鱼缸水</td></tr><tr><td>聚氨酯</td><td> Sigma - Aldrich公司</td><td> U2500 </td><td>剂量为2 g / kgloading </td></tr><tr><td>双管夜视镜</td><td>美国之夜光学</td><td> D - 221 </td><td></td></tr><tr><td>滤纸级4号</td><td>瓦特曼</td><td> 1004-090 </td><td></td></tr><tr><td>精细镊子，杜蒙，Biologie第5号，长11厘米</td><td>精细科学工具</td><td> 11295-10 </td></tr><tr><td>精细剪刀，弹簧加载，8毫米刀片，直</td><td>精细科学工具</td><td> 15025-10 </td><td></td></tR&gt; <tr><td> Neurobiotin </td><td>矢量</td><td> SP - 1120 </td><td> 0.5％ </td></tr><tr><td>链霉亲和共轭Alexa的488 </td><td> Invitrogen公司</td><td> S11223 </td><td> 2％ </td></tr><tr><td>葡聚糖罗丹明（高（&gt; 10,000）兆瓦） </td><td> Invitrogen公司</td><td> D1817 </td><td> 0.5％ </td></tr><tr><td> Vectashield安装介质</td><td>矢量</td><td> H - 1000 </td><td></td></tr><tr><td>蔡司510 META激光共聚焦显微镜</td><td>卡尔蔡司公司</td><td></td><td></td></tr><tr><td> LSM - 5图像浏览器3,2,0,115 </td><td>卡尔蔡司公司</td><td></td><td></td></tr><tr><td> ImageJ软件</td><td>国立卫生研究院</td><td></td><td></td></tr><tr><td> OriginPro 8.0 </td><td> OriginLab公司</td><td></td><td></td></tr></tbody></table>

cut-loading: a useful tool for examining the extent of gap junction tracer coupling between retinal neurons

除了化学突触传递，神经元缝隙连接连接也可以通过电突触传递迅速沟通。越来越多的证据表明，间隙连接不仅允许电流流动和细胞之间的相互关联或耦合的同步活动，但细胞之间的耦合强度或电气通信的有效性，可调制， 在很大程度上1,2。此外，内径大，许多缝隙连接通道（〜1.2 nm的）不仅允许电流流动，而且细胞内信号分子和细胞之间的相互关联的小代谢产物的扩散，使缝隙连接，也可调解代谢及化学通讯。同时电气耦合细胞记录和决定吨的神经元之间的差距及其神经递质及其他因素的调制交界通信的实力，可以研究他示踪分子的扩散，这是缝隙连接透水程度，但没有膜透水后，成单个细胞iontophoretic注射。然而，这些程序可以是非常困难的小somata完好的神经组织的神经元执行的。 在脊椎动物视网膜电突触和电气通信调制大量的研究已经进行，因为每五个视网膜神经类型是电连接缝隙连接3,4。越来越多的证据表明，昼夜（24小时）在光刺激视网膜和变化的时钟调节间隙连接耦合 3-8 。例如，最近的工作表明视网膜生物钟，减少白天的交界处耦合杆和锥感光细胞之间的差距，通过增加多巴胺D2受体激活，并通过降低D2受体的激活显着增加夜间杆锥耦合<suP&gt; 7,8。然而，不仅是这些研究非常困难的小somata完整的神经视网膜组织中神经元执行，但它可能很难充分控制在单一的视网膜神经细胞的电生理研究的光照条件，以避免在缝隙连接的光致变化电导。 在这里，我们提出了一个简单的方法确定不同的光照条件下，在白天和黑夜的不同时期的视网膜神经细胞之间的缝隙连接示踪剂耦合程度。这种切割加载技术是9-12的修改刮加载，这是基于对染料的装载和扩散通过开放的间隙连接通道。刮装载工程以及在培养的细胞，但不厚片，如完整的视网膜。削减加载技术已用于研究在完整的鱼和哺乳动物视网膜7，8,13的感光耦合，可以用来研究之间的耦合其他视网膜神经细胞，这里描述。

Watch this Scientific Journal Video about Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons at JoVE.com

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

描述一个简单方便的方法，以确定视网膜神经细胞之间的缝隙连接示踪剂耦合程度。这种技术，使一个完整的视网膜中的神经元之间的电突触的功能，不同的光照条件下，在白天和晚上不同时间的调查。

剪切载荷：一个有用​​的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

cut-loading-useful-tool-for-examining-extent-gap-junction-tracer

Research

JoVE Journal

Neuroscience

15.3K 次观看.  Ohio State University College of Medicine. 描述一个简单方便的方法，以确定视网膜神经细胞之间的缝隙连接示踪剂耦合程度。这种技术，使一个完整的视网膜中的神经元之间的电突触的功能，不同的光照条件下，在白天和晚上不同时间的调查。

视频: Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

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

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

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

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

科研

神经科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

抗体转染神经元作为一种工具来研究疾病的发病机制

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

一个吞噬含量：一个协议，以评估之间的中枢神经系统吞噬细胞和神经元的相互作用

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

小鼠视网膜冷冻节的制备,用于免疫性植物化学

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

A Standardized Pipeline for Examining Human Cerebellar Grey Matter Morphometry using Structural Magnetic Resonance Imaging

Multiple lines of research provide compelling evidence for a role of the cerebellum in a wide array of cognitive and affective functions, going far beyond its historical association with motor control. Structural and functional neuroimaging studies have further refined understanding of the functional neuroanatomy of the cerebellum beyond its anatomical divisions, highlighting the need for the examination of individual cerebellar subunits in healthy variability and neurological diseases. This paper presents a standardized pipeline for examining cerebellum grey matter morphometry that combines high-resolution, state-of-the-art approaches for optimized and automated cerebellum parcellation (Automatic Cerebellum Anatomical Parcellation using U-Net Locally Constrained Optimization; ACAPULCO) and voxel-based registration of the cerebellum (Spatially Unbiased Infra-tentorial Template; SUIT) for volumetric quantification. 
The pipeline has broad applicability to a range of neurological diseases and is fully automated, with manual intervention only required for quality control of the outputs. The pipeline is freely available, with substantial accompanying documentation, and can be run on Mac, Windows, and Linux operating systems. The pipeline is applied in a cohort of individuals with Friedreich ataxia (FRDA), and representative results, as well as recommendations on group-level inferential statistical analyses, are provided. This pipeline could facilitate reliability and reproducibility across the field, ultimately providing a powerful methodological approach for characterizing and tracking cerebellar structural changes in neurological diseases.

A standardized pipeline is presented for examining cerebellum grey matter morphometry. The pipeline combines high-resolution, state-of-the-art approaches for optimized and automated cerebellum parcellation and voxel-based registration of the cerebellum for volumetric quantification.

一种使用结构磁共振成像检查人小脑灰质形态测量的标准化管道

Multiple lines of research provide compelling evidence for a role of the cerebellum in a wide array of cognitive and affective functions, going far beyond ...

剪切载荷：一个有用​​的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度

剪切载荷：一个有用的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度