支持这项工作是由德意志研究联合会（SFB 655）和欧盟（ZF健康）。

1。注射液的制备<ol><li>使用细胞内在他们更有效地比正常的吗啉分子的体内吗啉。请参见制造商的信息资料（ 表1:试剂和材料）。 </li><li>用荧光示踪染料（ 如 CellTracker红CMTPX，Invitrogen公司）的可视化注射的准确性。这种染料在细胞代谢，并仅在细胞摄取后成为荧光。因此，这一步是至关重要的，以确定细胞，有效地由脑室微量注射对象。 </li><li>准备磷酸盐缓冲盐水（PBS）稀释吗啉溶液（137 mM氯化钠，2.7 mM的氯化钾，10mM的磷酸氢二钠，2 mM的KH 2 PO 4，pH值:7.4）。如果啉溶液稀释时，请务必使用PBS。 </li><li>准备注射10μl的混合，加入9微升的吗啉溶液和1μl的第ê细胞追踪染料（稀释或不稀释。股票啉溶液浓度为500微米。我们先前的研究表明，22几个不同剂量的吗啉-从50微米到500微米不等-从高效击倒在不同层次的结果击倒低表现量的表型）。 1微升溶液注射到斑马鱼脑都不够理想。 </li><li>搅拌均匀，存放在室温下，直到注射。 </li></ol> 2。注射的装置的下游<ol><li>准备玻璃注射毛细血管的使用针拉马。使用以下参数:供暖周期值:463，拉周期值:230，速度:1.5秒，时间:75毫秒。 </li><li>打开压力源，压力设置和调整到50磅或3.5酒吧。 </li><li>微量的设置调整如下:保持压力20磅，10磅弹出压力，周期值2.5和100毫秒范围内浇注。 </li><li>转环形照明和定位环在显微镜阶段。 </li><li>毛细管支架放置到适当的位置旁边的显微镜。 </li><li>插入到保持器的玻璃毛细管。调整喷射角度为45度。 </li><li>折断的玻璃毛细管的前端，用细镊子从孔校准的压力输出。所以必须进行测试成鱼水浸渍的针尖，并给予一个连续的压力脉冲。的所产生的气泡，应该只有一个行，这是最佳的压力的指示/孔口尺寸。请参阅该校准示范视频。 </li><li>装入玻璃毛细管注射溶液，无气泡的产生。 </li><li>施加压力，以除去玻璃毛细管的尖端和加载的注射液之间的空气。 </li></ol> 3。麻醉<ol><li>准备麻醉剂原液（0.1％MESAB - 乙基-米 - 氨基苯甲酸甲酯甲磺酸盐）。 </li><li>从坦克到塑料鼠笼与足够量的水或其他运输容器中取出所需数量的鱼。 </li><li>准备药物麻醉的解决方案，在一个小的塑料框中，然后通过鱼200毫升水混合，用5毫升的麻醉剂原液。麻醉剂的最终浓度为:0.0025％（体积/体积）。 </li><li>半麻醉剂填补了塑料培养皿。使用此注射菜。 </li><li>孵育麻醉药的鱼，直到鱼制胜。 </li></ol> 4。切口和脑室微量注射<ol><li>用鱼网从包装盒中取出的鱼，并把它放到培养皿。 </li><li>镊子和头部的方向轻轻按住鱼的方式，稍向下倾泻。这将有利于切口。 </li><li>生成头骨上使用的是30号刺针缝小。只能使用针头和不穿透多够进入大脑，因为这会造成脑组织的损害，表现为出血。 </li><li>持续按住鱼和通过切口部位插入尖端的玻璃毛细管。 </li><li>东方玻璃毛细管的尖端朝向端脑。避免接触的脑组织，因为这会产生组织损伤，也能防止注入溶液分散。 </li><li>注入的溶液。的液体注射后立即分散。 </li></ol> 5。注射精度的恢复和分析<ol><li>用新鲜的鱼水的塑料盒中取出鱼从注射设置和地点。 </li><li>允许恢复。 </li><li>重新麻醉与0.0025％MESAB的鱼简要鱼在荧光显微镜下检查。红色荧光是一个彻底的分散注射液的广泛分布的指标。 </li><li>把鱼放回去的塑料盒。 </li><li>塑料盒连接到t他循环系统，氧气的鱼和维持更长的时间。 </li><li>利用鱼的目的，如行为分析或牺牲的动物注射后准备组织标本作进一步分析（组织学染色，免疫组化等 ）所需的时间点。以下引用给成年斑马鱼脑:9,11,17-20,24-27如何分析的例子。最快的时间来观察一个有效的击倒（超过50％击倒）是12小时在我们的研究22。 </li></ol> 6。建议科学控制<ol><li>使用不匹配吗啉代分子是不应该造成任何表型作为阴性对照。使用一个标准吗啉分子或专门设计吗啉控制每一个感兴趣的基因。此外，使用溶剂（在这种情况下的PBS中），作为进一步的阴性对照，以评估非特异性作用或毒性MISM批次/控制啉注射。 </li><li>使用PCNA吗啉作为阳性对照先前描述22。这吗啉导致高效击倒在成年斑马鱼脑（75-95％击倒效率1天之内注射）22。 </li></ol>

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没有利益冲突的声明。

我们在这里描述的方法允许进入成年斑马鱼脑的吗啉的容易和快速的管理。我们已经证明，我们的注射方法有效地阻断基因的表达在心室肌细胞在神经元响应的功能性后果和结果。 有一些重要的点是持谨慎态度，同时执行脑室微量注射。例如，吗啉代分子的效果取决于所使用的浓度。这个浓度将由最终用户。我们建议开始与股票溶液（500微米），并进行系列稀释，最好预先测试注入胚胎，胚胎注射使用标准协议28-30。以前，我们获得了不同级别的拦截效率吗啉浓度的范围从50微米到500微米22。其次，节流孔的玻璃毛细管不应该被LARGe作为这将导致广泛的液体注射后涌入大脑。同样，开放必须不能过于狭窄，因为这将防止足够的注塑。人能够确定最佳的节流孔的压力的组合，通过空气泵入与水的陪替氏培养皿。所产生的气泡，应该是在一个单一的行，而不是在多个行。在视频中，我们证明这一点。第三，培育麻醉药的鱼是至关重要的。鱼不应存放时间超过2分钟，在麻醉药。这会妨碍注射后的回收率。第四，切口的位置是关键彻底分散注入的液体。在视顶盖心室区域中线以上大横向越走越窄。因此，实验者应该产生在颅骨缝接近中线，只是尾椎端脑区的颅骨内板覆盖。 脑室注射液的优点之一N（CVMI）方法是其快速性。 CVMI是一种快速的方法，用于测定基因的功能。此功能是重要和有益的，相比一代转基因株系的功能研究，一般需要几个月的时间。此外，CVMI导致注射的液体均匀分布的，因此提供了一个比较彻底的联络注射或电相比，操纵基因的活性。多个基因与CVMI，可以撞倒同时准备注射液混合含有多吗啉代寡核苷酸。 CVMI可用于一个给定的吗啉代寡核苷酸注入不同浓度的，因此可用于亚效等位基因表型分析。最后，这种注射范例不引起毒性或危及动物的生存。 其他类型的研究，如注射的调节肽，药物，质粒DNA或RNA的调节莫可能扩大的CVMI技术，lecules或其他物质有可能影响细胞生理状态的。测定分子组合和表演剂量反应分析是可能使用我们的方法，使学习低表现量的表型。凭借这些特性，CVMI被证明是一个快速简便的检测成年斑马鱼大脑中的表达研究，并开辟了快速筛选和功能分析。 组成的成年斑马鱼的大脑可以产生新的神经元沿整个rostrocaudal轴和外伤后还可以再生。这是哺乳动物的大脑，神经和有限，而如果在所有的再生能力差，形成了鲜明的对比。如此广泛的干细胞活性和休养的能力，使一个有用的模式生物斑马鱼了解分子程序所需的中枢神经系统的再生，这是目前很大程度上是未知的。因此，调查性向泽再生的分子基础brafish大脑是一个有趣的研究领域，这或许可以解释的根本区别，鱼类和哺乳动物的大脑是如何受伤后的反应，同时也赋予再生医学疗法在人类的途径。为了了解脊椎动物大脑的可塑性和再生的分子基础设施作为一个重要的研究领域，神经胶质细胞，因为它们是神经源性祖细胞3,8,20。因此，使用CVMI技术改变基因的斑马鱼大脑中的放射状胶质细胞的功能是在阐明如何鱼脑情侣祖活动高效的成年神经发生和再生反应。我们最近表明在成年斑马鱼脑24,26,27再生的神经发生响应的几个因素和信号通路的参与和要求，这些研究未能由使用CVMI方法。总体而言，从斑马鱼大脑中获得的知识，我们可以利用征收REGENErative哺乳动物的神经胶质细胞受伤的反应，从而帮助设计对人类神经系统疾病的临床治疗和急性伤害的能力。

成年神经发生的脊椎动物是一个共同的特点，但其患病率和效率程度不同与系统发育1,2,3,4,5,6。例如，成年哺乳动物的大脑中含有干细胞区域，很大程度上制约了前脑7,8，而硬骨鱼斑马鱼含有16个不同的干细胞领域和相关的神经源性区在其整个大脑4,5,9,10。这种变化可能反映了哺乳动物和斑马鱼之间的差距在维持干细胞和神经源性祖细胞的能力的机制。斑马鱼大脑中的神经元的终身能力也可以对斑马鱼脑的分子机制可能被利用为治疗中的应用，以解决人类神经退行性疾病的临床后果。 几种干细胞区的成年斑马鱼的大脑已经加以分析，它已被证明这些地区的心室面衬细胞作为祖细胞9,11-20。在斑马鱼端脑的详细分析，例如，确定了径向神经胶质细胞，划定该脑区域的心室表面神经源性祖细胞19，20。这也是如此，对其他地区，如小脑和视顶盖，心室位于神经上皮细胞神经源性输入16,21。为此，了解成年斑马鱼脑广泛神经源性能力的分子机制，需要操纵中的祖细胞的基因表达。 以前各种方法调节基因的功能在斑马鱼。这些措施包括生成条件的转基因株系，表达一个单一的蛋白质，质粒注射焦点耦合电或化学治疗所需的变种。我们之前设计了一个管理各种物质，包括吗啉代寡核苷酸或蛋白作为一种快速而有效的方式调节基因的功能在心室肌细胞的成年斑马鱼的前脑22使用脑室的显微注射到成年斑马鱼脑的方法。在我们的研究中，体内吗啉代被使用，因为他们的化学涉及吗啉分子共价连接到精氨酸丰富交纳的肽，从而可以有效的传递到感兴趣的细胞，而不需要额外的通透性，如电穿孔23。这将使一个彻底的定位所需的组织注射后，就像我们所观察到的成年斑马鱼端脑22。的体内吗啉使用，因此优于已有的方法，如电或局部注射DNA分子组织靶向。 在这里，我们直观地显示出我们如何执行此操作，并提供了一步一步的协议。我们先从解释准备注射液和鱼被注入，进行示范如何注射装置设置。我们提供描述脑室注射的方法，它涉及产生切口的吗啉代，用微量注射器在颅骨和注射。我们也阐述一个关键点需要通过优化或故障排除指南，说明他们在整个过程中要谨慎。

<table border="1">
	<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>CellTracker Red CMTPX</td>
 <td>Life Technologies, Invitrogen</td>
 <td>C34552</td>
 <td>Use at 1:100 dilution for measuring the injection accuracy</td>
 </tr>
 <tr>
 <td>MESAB (ethyl-m-aminobenzoate methanesulphonate)</td>
 <td>Sigma-Aldrich</td>
 <td>A5040</td>
 <td>For anesthetizing the fish</td>
 </tr>
 <tr>
 <td>Petri dishes</td>
 <td>Sarstedt</td>
 <td>821,472</td>
 <td>For handling the fish during injection and imaging</td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Life Technologies, GIBCO</td>
 <td>10010-056</td>
 <td>As sterile dilution medium</td>
 </tr>
 <tr>
 <td>Vivo morpholinos</td>
 <td>Gene Tools Inc.</td>
 <td>Customized</td>
 <td>More info on vivo morpholinos: <a href="http://www.gene-tools.com" target="_blank">www.gene-tools.com</a></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Barbed-end needle</td>
 <td>Becton-Dickinson</td>
 <td>305178</td>
 <td>To generate the incision in the skull</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Olympus, Leica, Zeiss </td>
 <td>Varies with the manufacturer</td>
 <td>Part of the whole injection setup</td>
 </tr>
 <tr>
 <td>Dumont Tweezers</td>
 <td>World Precision Instruments</td>
 <td>501985</td>
 <td>To snap off the tip of the glass capillary</td>
 </tr>
 <tr>
 <td>Fluorescence camera</td>
 <td>Zeiss, Nikon, Leica</td>
 <td>Varies with the manufacturer</td>
 <td>To visualize the fluorescence after injection</td>
 </tr>
 <tr>
 <td>Gillies Dissecting Forceps</td>
 <td>World Precision Instruments</td>
 <td>501265</td>
 <td>To hold the fish in position</td>
 </tr>
 <tr>
 <td>Glass injection capillaries</td>
 <td>World Precision Instruments </td>
 <td>TWF10 </td>
 <td>For microinjection</td>
 </tr>
 <tr>
 <td>PicoNozzle kit (microinjector holder)</td>
 <td>World Precision Instruments</td>
 <td>5430-12</td>
 <td>For microinjection</td>
 </tr>
 <tr>
 <td>Pneumatic PicoPump (microinjector)</td>
 <td>World Precision Instruments</td>
 <td>SYS-PV820 </td>
 <td>For microinjection</td>
 </tr>
 <tr>
 <td>Ring illuminator; Ring Light Guide</td>
 <td>Parkland Scientific</td>
 <td>ILL-RLG </td>
 <td>For illuminating the specimen</td>
 </tr>
	</tbody>
 </table>

micromanipulation of gene expression in the adult zebrafish brain using cerebroventricular microinjection of morpholino oligonucleotides

操纵基因表达的组织需要进行功能研究。在本文中，我们证明脑室微量注射（CVMI）的技术作为一种手段来调节基因表达的成年斑马鱼脑。物质通过使用CVMI，可以施用到脑室流体和彻底分布沿rostrocaudal的轴的大脑。我们尤其侧重于使用的吗啉代反义寡核苷酸，是强有力的工具，撞倒体内基因表达。在我们的方法中，当应用，吗啉分子采取了由内壁细胞的心室面。这些细胞的放射状胶质细胞，神经源性祖细胞作为。因此，撞倒放射状胶质细胞的基因表达是最重要的分析斑马鱼的的广泛神经发生反应，也将提供洞察脊椎动物如何维持成人neurogen的ESIS响应。这样的理解，也将有助于在人类神经退行性疾病的临床应用和中枢神经系统的再生的努力。因此，我们提出了一种快速而有效的方式来改变基因的表达在成年斑马鱼的前脑和神经反应的脑室微量注射的方法。我们还提供故障排除提示和其他有用的信息，如何开展的CVMI程序。

脑室微量注射导致注入的解决方案广泛分布使用图1中描绘的脑室微量注射的工作流程和技术方案，吗啉代寡核苷酸给药到成年斑马鱼的大脑。一个准确的注入协议，将导致整个大脑，高效的目标靠近心室表面（ 图2A）的细胞分散液注入液体。注射玻璃毛细管刺穿的脑组织在注入点的（ 图2B），将有一个致密的荧光信号。如果注入的量是不够的，将观察到微弱的荧光信号（ 图2C）。 可用于基因表达于成年斑马鱼脑心室区域撞倒脑室微量注射我们已经表明日在体内吗啉代可以穿透几个细胞直径的心室面内，因此可以有效地针对心室肌细胞（ 图3A，3B）。吗啉注射液可以拦截基因表达的，因为它会减轻相应的蛋白质生产（例如增殖细胞核抗原所示， 图3C，图3D）。之前我们已经证实，使用这种技术的基因敲除导致在成人神经（ 图3E，3F）或再生反应（ 图3G，3H）的功能的后果。我们表明，注射后约12小时的吗啉代22达到高效的拦截范围（超过50％击倒）。我们还观察到，有效的击倒期间，直到约5天注射后的PCNA蛋白22。超过70％的击倒，直到3天后注射液22。击倒曲线依赖Øn的水平表达的内源性蛋白和吗啉代分子的效率，还必须确定每一个基因被实验者。在我们的研究中，我们没有看到的CVMI程序危及生存的动物，可能是因为管理啉分子是局部的，不会引起全身中毒。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50415/50415fig1highres.jpg" src="/files/ftp_upload/50415/50415fig1.jpg" /> 图1。脑室注射技术的示意图。 （A）注射液是从背视顶盖的水平（OT 2）成年斑马鱼（1）。作一个切口，用30号针（3）光纤顶盖颅骨内板。使用玻璃毛细管，吗啉代分子注入脑室液（4）。一个彻底的分散是一个（E）瞬间长足的进步（5）（B）所示的套管，它的带刺端在高倍率（C）前切口。针的方向（D）的切口圈，由虚线概述。位置如图所示，并进行喷射的喷射毛细管。所有的面板都进行了调整，从文献22。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50415/50415fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/50415/50415fig2.jpg" /> 图2。注入效率和准确性。比较（A）良好的注塑针对靠近心室表面细胞。 （B）如果玻璃毛细管插入进入脑组织，这将产生一个位于中心的荧光信号。 （C </s仲&gt;）如果喷射量是低的，这会导致弱的荧光信号。 <img alt="图3" fo:content-width="5.5in" fo:src="/files/ftp_upload/50415/50415fig3highres.jpg" src="/files/ftp_upload/50415/50415fig3.jpg" /> 图3。吗啉代渗透，（A）的效率基因击倒和功能的后果。GFP免疫染色吗啉代注射的Tg（H2A:GFP）转基因品系显示GFP阳性细胞核的控制端脑截面。 （B）绿色荧光蛋白免疫组化染色端脑截面GFP反义吗啉注射TG（H2A:GFP）转基因株系GFP阳性核。注意心室区域的GFP信号的差异。 V:心室。 （C），增殖细胞核抗原免疫组化控制啉注入大脑PCNA阳性细胞广泛分布。 （ <strong&gt; D）PCNA免疫组化PCNA反义吗啉注入大脑PCNA阳性细胞显着减少的金额，表示CVMI可以敲除内源性基因。 （E）BrdU和HUC / D免疫组化控制吗啉注入大脑新生神经元前22后的BrdU脉冲追踪实验来确定。 （F）BrdU和HUC / D免疫组化PCNA反义吗啉注入大脑新生神经元前22后的BrdU脉冲追踪实验来确定。 （G）敲减PCNA导致新生神经元的产生显着降低，表明CVMI可用于拦截内源性基因，产生功能的后果。所有的面板摘自文献22 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50415/50415fig3large.jpg" target="_blank">点击此处查看大图</a> 。

Watch this Scientific Journal Video about Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides at JoVE.com

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

在这篇文章中，我们展示了一个使用吗啉反义寡核苷酸的成年斑马鱼端脑心室肌细胞的基因表达的方法操纵。我们将此方法作为一个高效，快速的协议，该协议可用于在成年脊椎动物脑的功能性研究。

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

micromanipulation-gene-expression-adult-zebrafish-brain-using

Nanyang Technological University

Research

JoVE Journal

Neuroscience

19.2K 次观看.  Cluster of Excellence (CRTD) and Biotechnology Center (BIOTEC) of the Technische Universität Dresden. 在这篇文章中，我们展示了一个使用吗啉反义寡核苷酸的成年斑马鱼端脑心室肌细胞的基因表达的方法操纵。我们将此方法作为一个高效，快速的协议，该协议可用于在成年脊椎动物脑的功能性研究。

视频: Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二:外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

科研

神经科学

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...