This work was funded by the DFG (SFB 779 TPB8, Kr1879/3-1 MRK), DIP grant (MRK), EU FP7 MC-ITN NPlast (MRK), Center for Behavioral Brain Sciences (CBBS, Sahsen-Anhalt), (AK and SB), MM is a recipient of European Molecular Biology Organization (EMBO) Long-Term Fellowship (EMBO ALTF 884-2011) and Marie-Curie IEF.

从成年大鼠脑1。制备急性海马脑片<ol><li>麻醉大鼠异氟醚。注意:执行使用封闭exicator的过程中，不要吸入异氟醚。确保动物完全麻醉。 </li><li>杀头的大鼠，迅速 ​​隔离大脑，并沉浸在precarbonated（95％O 2/5％CO 2的混合气体）冰冷Gey中的溶液（组成MM:130氯化钠，4.9氯化钾，1.5氯化钙2·2H 2 O ，0.3硫酸镁4·2H 2 O，11氯化镁2·6H 2 O，0.23 KH 2 PO 4，0.8的Na 2 HPO 4·7H 2 O，5个葡萄糖·H 2 O，25 HEPES，22 碳酸氢钠 ，pH值7.32） 30分钟1,2,10,16。 </li><li>取下内嗅皮层，小脑和组成部分。与正中矢状切分出皮质半球，然后将每个半球倒在其内侧表面。此后使50-70°切割（50-70°横向）沿着每个半球13,14的背缘。 </li><li>胶水每个半球的切片系统的切片平台上的新鲜切割面。该平台应涵盖precarbogenated冰冷Gey中的解决方案。 </li><li>切350微米50-70°横切片由前至后一边用调整，以减少Z轴振荡àvibratome。海马结构，subicular和内嗅皮质，以及它们位于背侧海马的皮层将用于实验13,14的条带的一部分。 </li><li>转移海马为马蹄形，并浸没式孵化器和孵化至少2小时，在32℃，carbogenated人工脑脊液（学联含MM:110氯化钠，氯化钾2.5，2.5氯化钙2·2H 2 O，1.5硫酸镁4·2H 2 O，1.24 KH 2 PO 4，10葡萄糖·H 2O，27.4 碳酸氢钠 ，pH值7.3），1,2,10,16。 </li></ol> 2，定位电极的基线记录，和LTP的诱导<ol><li>海马切片传送到​​安装在显微镜下浸没式录音室。灌注（6-7毫升/分钟）与carbogenated ACSF中至少30分钟，在32℃下。 </li><li>准备玻璃毛细管微电极填充学联（尖阻力为3-5MΩ）。 </li><li>放置一个玻璃微电极填充ACSF中的CA1谢弗抵押纤维的刺激和在海马CA1辐射层为fEPSP记录1,2,10（ 图2）。电极之间的距离应为大约300微米。 </li><li>由谢弗抵押纤维与双相方波电流脉冲（200毫秒/极性）的刺激的范围3-4五唤起场兴奋性突触后电位（fEPSPs）加强</li><li>通过测量INP执行的最大刺激试验UT输出关系和定义的刺激强度作为获得最大fEPSP斜率值的40％，并保持它在整个实验常数。 </li><li>开始基线记录的最大刺激试验后至少15分钟。测量的响应来测试刺激整个实验过程中，每分钟。执行基线记录用低频刺激。 </li><li>记录基线为至少30分钟。 </li><li>对于晚期LTP的诱导采用高频100赫兹强直电刺激由三个1秒刺激的列车在100赫兹有5分钟intertrain间隔。为了增加在CA1区5微米荷包牡丹碱刺激的字段可以洗2分钟强直电刺激之前和最后强直电刺激后，应立即被淘汰。 </li></ol> 3，收集片的LTP和快速冷冻的诱导后<ol><li>停止LTP记录2分钟或强直性刺激的三列火车诱发晚期LTP后30分钟。 </li><lI&gt;取出电极，快速切片转移到precold金属平台上放置干冰。重复该过程控制切片保持在U型孵化器。注意:使用手套，同时用干冰工作。 </li><li>收集在1.5ml试管和存储每片在-80℃。 </li></ol>从海马CA1区4​​核的分离富集馏分<ol><li>取1.5毫升的Eppendorf管中冷冻切片从-80℃并保持在冰上。 </li><li>加入0.5ml的新鲜冷的TBS缓冲液含有蛋白酶（PI）和磷酸酶（PS）的抑制剂，进入管毫升。孵育2-3分钟，并传输到立体。注意:使用防护手套和实验室外套，同时采用PI和PS工作。 </li><li>解剖用两针的海马CA1锥体层区域。用一针，用于保持在体视显微镜和第二针用于切割CA1区下的切片。 </li><li>收集解剖CA1区域从5片（每组）到新的1.5毫升试管中含有50μl的裂解缓冲液（1×含以mM低渗裂解缓冲液:10 HEPES，1.5的MgCl 2，10的KCl，pH值7.9，用PI和PS）。请注意，此材料用量将足以运行2-3免疫印迹。 </li><li>均质化通过小心移液上下收集的组织。用200微升吸管。在冰上孵育的裂解液为5-7分钟以使细胞溶胀。 </li><li>取2微升的裂解液，滴在显微镜载玻片，并可视化明视场显微镜下的细胞的肿胀。与细胞的正常肿胀的细胞核显示为圆形完整结构。 </li><li>收集20微升样品到新的1.5 ml管的"匀浆分数"。添加8微升变性4×SDS上样缓冲液中。 </li><li>离心剩余的溶胞产物在11000 rpm离心1分钟。 </li><li>小心地从顶部（&#39;胞浆部分"）收集上清液，TRansfer到一个新的管中，加入20微升的4倍上样缓冲液。 </li><li>悬浮颗粒在60微升低渗缓冲液加入20μl4X采样缓冲区。该馏分被称为一个"核浓缩部分&#39;。 </li><li>匀浆，胞浆和核富含分可以储存在-20℃或-80℃，以后免疫。 </li></ol> 5，半定量免疫印迹的突触核蛋白质<ol><li>解冻的样品和在95℃下煮沸5分钟。 </li><li>采取从每个级分5微升，并确定由氨基黑测试或BCA测试中的蛋白质浓度。 </li><li>装载于SDS-PAGE电泳从匀浆等量的蛋白样品，细胞质和细胞核富集馏分。从控制和LTP切片样品应放置在同一凝胶的直接比较。 </li><li>执行标准的西方印迹程序（湿传递推荐）。 </li><li>探索与T膜他抗体的选择。随后，同样的印迹（或并行运行同一样品）可以探测与细胞质标记 - 神经元特异性enolase2（NSE2）和核标志 - 的NeuN和肌动蛋白抗体作为内参。 </li></ol> 6，数据分析<ol><li> LTP的诱导效率可以通过Clampfit软件进行分析。基线记录的平均坡度使用强直电刺激后的山坡上双因素方差分析，P比较&lt;0.05为差异有显著不同。该fEPSP斜率值在图中表示为均值±标准差</li><li>对于免疫印迹定量，无论是扫描的放射自显影胶片和分析蛋白条带通过的ImageJ或荧光带集成密度与LICOR系统。免疫反应性条带的数值应被归一化的负载和印迹控制。对于控制的比较和LTP的用户组nonparametrical采用Mann-Whitney U检验可能使用。 </li></ol><table border="0"CELLPADDING ="0"CELLSPACING ="0"&gt; <tbody><tr><td> 缓冲区的命名 </td><td> 试剂 </td><td> 浓度（mM） </td><td> 金额 </td><td> 评论/说明 </td></tr><tr><td> Gey中的溶液 （pH值:7.3〜7.4） </td><td>氯化钠</td><td> 130 </td><td> 7.6克</td><td>无菌过滤</td></tr><tr><td>千毫升</td><td>氯化钾</td><td> 4.9 </td><td> 0.37克</td><td></td></tr><tr><td></td><td>氯化钙2·2H 2 O </td><td> 1.5 </td><td> 0.22克</td><td></td></tr><tr><td></td><td>硫酸镁4·2H 2 O </td><td> 0.3 </td><td> 0.0739克</td><td></td></tr><tr><td></td><td>氯化镁2·6H 2 O </td><td> 11 </td><td> 2.24克</td><td></td></tr><tr><td></td><td> KH 2 PO 4的</td><td> 0.23 </td><td> 0.0313克</td><td></td></tr><tr><td></td><td>的Na 2 HPO 4·7H 2 O </td><td> 0.8 </td><td> 0.2145克</td><td></td></tr><tr><td></td><td>葡萄糖·H 2 O </td><td> 5 </td><td> 0.9909克</td><td></td></tr><tr><td></td><td> HEPES </td><td> 25 </td><td> 5.96克</td><td></td></tr><tr><td></td><td> 碳酸氢钠 </td><td> 22 </td><td> 1.85克</td><td></td></tr><tr><td> 学联 （pH值:7.3〜7.4） </td><td>氯化钠</td><td> 110 </td><td> 6.428克</td><td>无菌过滤</td></tr><tr><td>千毫升</td><td>氯化钾</td><td> 2.5 </td><td> 0.1865克</td><td></td></tr><tr><td></td><td>氯化钙2·2H 2 O </td><td> 2.5 </td><td> 0.368克</td><td></td></tr><tr><td></td><td>硫酸镁4·2H 2 O </td><td> 1.5 </td><td> 0.370克</tD&gt; <td></td></tr><tr><td></td><td> KH 2 PO 4的</td><td> 1.24 </td><td> 0.169克</td><td></td></tr><tr><td></td><td>葡萄糖·H 2 O </td><td> 10 </td><td> 1.9817克</td><td></td></tr><tr><td></td><td> 碳酸氢钠 </td><td> 27.4 </td><td> 2.3克</td><td></td></tr><tr><td> 1X低渗缓冲液 （pH值:7.9） </td><td> HEPES </td><td> 10 </td><td> 0.23克</td><td></td></tr><tr><td>百毫升</td><td>氯化镁2·6H 2 O </td><td> 1.5 </td><td> 0.0304克</td><td></td></tr><tr><td></td><td>氯化钾</td><td> 10 </td><td> 0.07455克</td><td></td></tr></tbody></table> 表1缓冲器。

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The authors have nothing to disclose.

在上述协议中所述的步骤提供指导如何准备从青年或成年大鼠海马急性切片，诱导和记录LTP，迅速解剖切片的刺激区域，并准备核浓缩部分的研究活动相关的蛋白质动力学。这种方法源于对彼此独立地使用几种不同的方法组合。我们优化的工作流程，并为初学者建立自己的实验后，LTP的诱导，研究蛋白质的亚细胞再分配提供足够的细节。作为一个例子，我们证明了LTP的晚期形式诱导S180的核积累磷...

突触N-甲基-D-天冬氨酸受体（NMDA受体）发挥在突触可塑性和细胞存活信号而激活突触外NMDA受体的可触发的神经变性和细胞死亡的关键作用。这些变化依赖于严密控制/受规管活动相关的基因表达，因此需要激活突触或树突和细胞核7之间不断的沟通。在MAP激酶ERK1 / 2的突触NMDA受体的信号的下游效应，并参与NMDA受体活化诱导的基因表达，而经由突触外NMDA受体信号没有或ERK1 / 2的活性8,11的抑制作用。 有迹象表明，已经显示出远端树突和细胞核之间穿梭蛋白的数量。许多这些蛋白质含有核定位信号，并正在积极沿着在动力蛋白和importin依赖性微管运输到核6,9。 InterestinglY，一些使者只是过境到细胞核针对特定突触的刺激。例如，环AMP应答元件结合蛋白2（CREB2）的逆行运输，通过化学股份有限公司但不LTP 12诱导。本地化的NMDA受体依赖的突触刺激驱动CREB调节转录共激活因子（CRTC1）进入细胞核，易位过程，其涉及在长期海马可塑性4。它最近的研究表明，该蛋白信使雅各布转位到后两个核，突触和突触外NMDA受体的激活和调节CREB依赖性基因转录的5。信号的突触和突触外原点编码雅各的翻译后修饰。突触活动引起雅各ERK1 / 2磷酸化依赖于在位置180（pJacobS 180）的一个关键丝氨酸这是一个必要的后续易位初级海马文化的核心。此外，我n个急性海马pJacobS 180转位到谢弗抵押LTP后核CA1区神经元而不是公司咯 。 pS180雅各导致的可塑性相关的基因表达增加，这基因表达反馈到突触功能。形成鲜明对比，雅各布该易位至细胞核后突触外NMDA受体活化不磷酸化在Ser180和可能具有不同的蛋白复合物有关在核中引起"CREB切断"和突触触头10回缩。 对突触核蛋白质的信使核进口的大多数已发表的研究中分离的神经细胞原代培养工作已经完成。因此，这将是有趣的，如果这样的调查结果可以复制使用海马生理更多的相关条件下神经元的连接和功能更好的保存。在这里，我们提出了一个优化的协议，以评估LTP-DE通过免疫印迹侧基的蛋白质信使核转。这个方法也适合于分析中的粗核级分的蛋白质的活性依赖的磷酸化。具体来说，目前的协议包括制备急性海马CA1区片，感应和记录的LTP。接着，CA1区用显微镜解剖，分离出刺激的区域。我们结合和修饰赵和他的同事17引入的更改提供CellLytic核抽提试剂盒的协议核隔离。优化过程包括解剖海马CA1区的低渗缓冲液裂解使细胞肿胀，核释放。细胞裂解和细胞核形态可以通过显微镜检查来确定。铀浓缩是一个短离心步骤来实现的。免疫印迹分析用抗体的NeuN和NSE2，核或细胞质级分的特异性标记物，表明该方法可以被用作快速和可重复的协议来隔离这些亚细胞组分，并研究样蛋白的磷酸化非常不稳定的翻译后修饰。此外，此方法是有利的，对于小的组织样本来自海马切片的CA1区解剖区域导出，并且可以组合使用，以海马切片的免疫组化分析。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>CED 1401 AD/DA converter</td><td>Cambridge Electronics Design, UK</td><td></td><td></td></tr><tr><td>&lt;a href=&quot;http://www.iciba.com/stereomicroscope&quot;&gt;Stereomicroscope&lt;/a&gt;</td><td>Leica S4E, Germany</td><td></td><td></td></tr><tr><td>Vibrotome</td><td>Leica VT1000S, Germany</td><td></td><td></td></tr><tr><td>Isolated pulse stimulator</td><td>A-M Systems, USA</td><td></td><td>Model 2100</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>SDS-PAGE system</td><td>Biorad</td><td></td><td></td></tr><tr><td>Cooler</td><td>Julabo</td><td></td><td></td></tr><tr><td>Bright field Microscope</td><td>Nikon Eclipse TS100</td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td>Thermo Electron Corporation</td><td></td><td></td></tr><tr><td>Micromanipulator</td><td>Luigs &amp;amp; Neumann, SM-5, Germany</td><td></td><td></td></tr><tr><td>Blotting chamber and electric power supplier</td><td>Hoefer Scientific Instruments, San Francisco, CA</td><td></td><td></td></tr><tr><td>Submerged type recording chamber</td><td>custom made</td><td></td><td></td></tr><tr><td>U-shape and submerged type incubator&amp;nbsp;</td><td>custom made</td><td></td><td></td></tr><tr><td>Small surgical scissors</td><td></td><td></td><td></td></tr><tr><td>Scalpel</td><td></td><td></td><td></td></tr><tr><td>Thin spatula</td><td></td><td></td><td></td></tr><tr><td>Plastic Pasteur pipette</td><td></td><td></td><td></td></tr><tr><td>Plastic culture dish&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Bunsen beaker</td><td></td><td></td><td></td></tr><tr><td>Syringe</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>NaCl</td><td>Roth</td><td>Art.-Nr.3957.1</td><td>&amp;ge;99.5%, p.a., ACS, ISO</td></tr><tr><td>KCl</td><td>Roth</td><td>Art.-Nr.6781.1</td><td>&amp;ge;99.5%, p.a., ACS, ISO</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>1.02382.0500</td><td>pro analysis</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>5886.05</td><td>pro analysis</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>AppliChem</td><td>CAS-NO: 7791-18-6; EC-NO:2320946</td><td>for molecular biology</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>12034.025</td><td>for molecular biology</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>1.06574.1000</td><td>extra pure</td></tr><tr><td>Glucose&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Roth</td><td>Art.-Nr.6887.1</td><td>for molecular biology</td></tr><tr><td>HEPES</td><td>Roth</td><td>Art.-Nr.9105.4</td><td>PUFFERAN, &amp;ge;99.5%, p.a.</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Merck</td><td>1.06329.1000</td><td>pro analysis</td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche</td><td></td><td></td></tr><tr><td>Phosphostop</td><td>Roche</td><td></td><td></td></tr><tr><td>Bicuculline</td><td>Tocris Bioscience</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Baxter</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Antibodies&lt;/strong&gt;</td></tr><tr><td>Primary antibodies</td><td>Company</td><td>Catalog Number</td><td>Species</td></tr><tr><td>pJac-s180</td><td>Biogenes</td><td></td><td>rabbit (diluted 1:100)</td></tr><tr><td>NeuN</td><td>Milipore</td><td>MAB377</td><td>mouse (diluted 1:1,000)</td></tr><tr><td>NSE</td><td>Cell Signaling</td><td>D20H2</td><td>rabbit (diluted 1:1,000)</td></tr><tr><td>Beta-actin</td><td>Sigma</td><td>A-5441</td><td>mouse (diluted 1:5,000)</td></tr><tr><td>Secondary antibodies</td><td>Company</td><td>Catalog Number</td><td>Species</td></tr><tr><td>IgG HRP conjugated</td><td>DAKO</td><td></td><td>goat anti-mouse (1:5,000)</td></tr><tr><td>IgG HRP conjugated</td><td>NEB</td><td></td><td>goat anti-rabbit (1:5,000)</td></tr></tbody></table>

isolation of ca1 nuclear enriched fractions from hippocampal slices to study activity-dependent nuclear import of synapto-nuclear messenger proteins

学活性依赖的蛋白表达，细胞内转位，或磷酸化是必不可少的理解突触可塑性的潜在的细胞机制。长时程增强（LTP）和诱导的急性海马长时程抑制（LTD）被广泛地接受为学习和记忆的细胞模型。有迹象表明，使用活细胞成像和免疫组化的方法来可视化活动相关的蛋白质动力学大量的研究。然而，这些方法依赖于抗体的免疫细胞化学在单个神经元的荧光标记蛋白或过表达的适宜性。蛋白质的免疫印迹是一种替代方法提供的结果，独立确认。在制备由个别tetanized海马切片的亚细胞级分的第一限制因素是材料的低量。二，处理程序是至关重要的，因为升甚至很短，小的操作艾尔文片可能会引起一定的激活信号级联。在这里，我们描述，以便获得足够纯度的核浓缩部分的足够量的从大鼠脑急性海马切片的CA1区的优化工作流程。作为一个代表性的例子中，我们表明，突触核蛋白质的信使的ERK1 / 2磷酸化形式雅各积极进入细胞核后，诱导LTP，并且可以从CA1区神经元核浓缩部分进行检测。

之前我们已经表明，突触核蛋白质的信使雅各下诱导LTP，但不有限公司1个聚集在细胞核内。此外，刺激突触后转雅各需要激活蛋白激酶ERK1 / 2磷酸化雅各在Ser180（ 图1）。磷酸雅各易位至细胞核中的importin依赖性和磷酸化状态可以被保存在延长的时间周期由协会与中间丝αinternexin15（ 图1）。雅各的磷酸化状态是对的活性依赖性基因和细胞存活的表达很重要...

Watch this Scientific Journal Video about Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins at JoVE.c

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

我们提供了用于诱导的长时程增强，在海马CA1区和核富集级分的从片的tetanized区域的随后分离的详细协议。这种方法可以被用来确定活性依赖的核蛋白质导入在学习和记忆的细胞模型。

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

isolation-ca1-nuclear-enriched-fractions-from-hippocampal-slices-to

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

12.0K 次观看.  Leibniz Institute for Neurobiology. 我们提供了用于诱导的长时程增强，在海马CA1区和核富集级分的从片的tetanized区域的随后分离的详细协议。这种方法可以被用来确定活性依赖的核蛋白质导入在学习和记忆的细胞模型。 

视频: Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

科研

神经科学

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

使用急性海马脑片啮齿动物突触标记/捕获和交叉捕获的研究

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham &#34;no seizure&#34; rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

大鼠电癫痫发作与海马的分离检测突触后密度蛋白的变化

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described here identifies the basic building blocks of this circuitry. The in-depth studies of cortical regions will ultimately provide other scientists with the circuit components needed for an understanding of how the brain acquires, processes and stores information and what goes wrong in disease, while the electrophysiological and morphological data are widely used by computational neuroscientists in the construction of model networks that explore information processing. The protocol outlined here describes how biocytin-filled cells recorded in the CA2 region of the hippocampus are recovered and then reconstructed in&#160;3D. Additionally, the protocol describes the demonstration of calcium binding protein or peptide content in recorded interneurons.

The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied.

填充海马 ca2 间神经元的生物环素恢复与三维重建

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

鼠脑水平海马片

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.

We have developed a reliable method of selective plasma membrane permeabilization of primary mouse cortical neurons for high content automated analysis of neuronal nucleocytoplasmic transport.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing ...

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

亚细胞分离用于从小鼠脑中分离突触成分

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological ...

A Technique to Isolate Nuclei from Non-neuronal Cells in the Hippocampal Dentate Gyrus

Homogenize the tissue with 10 strokes of the loose A pestle, followed by 15 strokes of the tight B pestle. Transfer the homogenate into a pre-chilled 15-milliliter tube. Rinse the Dounce homogenizer with 1 milliliter of cold HB, and combine it with the same tube.
Add 3 milliliters of HB to the 15-milliliter tube, and incubate for 5 minutes on ice. Mix this nuclei suspension twice by inverting the tube gently. Using 0.5 milliliters of HB, pre-wet the 70-micrometer strainer cap placed over a 50-milliliter test tube. Then, strain the nuclei suspension before washing the cell strainer with 0.5 milliliters of HB.
Next, remove the cell strainer, and centrifuge the test tube in a swing bucket centrifuge at 500 g for 5 minutes at 4 degrees Celsius. Discard the supernatant. Gently resuspend the pellet in 4 milliliters of HB using a P1000 pipette.
After five minutes of incubation on ice, centrifuge the suspension at 500 g for 10 minutes at 4 degrees Celsius. Discard the supernatant, and resuspend the pellet in 3 milliliters of wash media. Use 0.5 milliliters of wash media to pre-wet a 35-micrometer strainer cap over a 50-milliliter test tube. Then, strain the nuclei suspension by pipetting 0.5 milliliters of suspension at a time using P1000 pipette.
After washing the strainer cap with 0.5 milliliters of wash media, place the tube on ice. Transfer the filtrate to a new 15-milliliter tube, and centrifuge it for 5 minutes and 4 degrees Celsius at 500 g. Discard the supernatant, and resuspend the pellet in 3 milliliters of wash media.
Repeat the centrifugation, and resuspend the pellet in 1 milliliter of wash media with the mouse anti-Neu and AlexaFlour488-conjugated antibody and 1 microgram per milliliter DAPI. Incubate the reaction for 45 minutes on ice in the dark.
For Fluorescence Activated Nuclei Sorting or FANS, transfer the immunostained nuclei suspension to a 5-milliliter test tube, and place it on ice until the start of the flow cytometry procedure.
Vortex the samples for three seconds with mild intensity before placing the tubes into the FACS instrument.
To acquire the data from stain nuclei suspension, set the gates in DAPI height and the DAPI area to exclude the cell debris and the aggregated nuclei. Then, set the gates in the log side-scatter or SSC area and the log forward-scatter or FSC area to separate the single nuclei from the remaining DAPI-stained aggregate or cell debris.
Then, isolate the NeuNAF488-negative population by setting the gates for the anti-NeuNAF488 and FSC area.
After the analysis, sort the anti-NeuNAF488-negative population in a 1.5-milliliter collection tube filled with 50 microliters of wash media using the gating strategy.