这项工作是由马克斯·普朗克研究所精神病学和欧盟的玛丽·居里的初始培训网络（如心合同238665）。

1。由早期的生活逆境的编程母婴分离（MS）进行早期生活压力（ELS）的诱导由定时孕妇C57BL/6N小鼠（产后一天出生当天（P0））提供的幼仔。 <ol><li>个别窝放置在清洁的笼子里每天3小时（加热垫）从P1-10。 </li><li>控制（非ELS）的幼崽留在产妇巢整个原状。 </li><li> （P21蛋白），断奶后的时间，他们安置在性别相匹配的组标准的实验室动物的住房条件下（每笼3-5只）保持与他们的母亲，直到幼崽。 </li></ol> 2。脑组织中分离和解剖<ol><li>小鼠被杀害在颈椎脱位注意所需的年龄，因为该协议涉及的应力范式，没有麻醉前给予颈椎错位，以避免干扰正常的应激激素的生理调节。头骨被打开，大脑小心取下，并立即冻结单元，沉浸在异戊烷干冰，之前被储存在-80°C </li><li>的大脑cryosectioned（厚度为10微米），路段安装对Superfrost玻片，并保持在-20°C参考标准鼠标立体地图集（如Paxinos 6）是用来验证解剖的精度，以确保该地区的利益（如在室旁核神经元-下丘脑室旁核-延髓PVN内，囟门的水平开始收集部分列入-0.75至-0.85）。 </li><li>甲酚紫染色部分，以便识别不同的大脑结构。拳（0.8毫米）的利益的地区（如PVN内），得到了局部显微。 </li></ol> 3。来自脑拳核酸提取一个优化的协议，同时提取DNA和RNA的微小的神经解剖学定义的大脑章亚硫酸氢钠和基因表达分析的离子描述7。 注：考虑到RNA是不到GTC的缓冲区中的DNA的稳定，我们建议先处理的RNA。用于DNA纯化匀浆可以在这段时间里保持在室温（RT）。 <ol><li>在使用400μL的异硫氰酸胍（GTC的）缓冲区（异硫氰酸胍4.5米，2％的N-lauroylsarcosine，50 mM EDTA的pH值8.25毫米的Tris-HCl pH值7.5，0.1 M的β-巯基乙醇，0.2％，消泡剂吸管和vortexer的均质拳一）在室温下，多次通过皮下注射针筒（29G）（ 图2）。 </li><li>将等份分割裂解物;可同时提取RNA和DNA或单独，取决于在特定的实验需要。 </li><li> RNA纯化，添加量的1/10，醋酸钠，1的体积AquaPhenol（Appligene）（pH值4）和1/2体积的氯仿：异戊酯（24:1）裂解物。每一步后大力涡和在冰上孵育10分钟，离心（20分钟在10,000 g 4℃）;添加70％乙醇的等量aequous阶段。 </li><li>混合物转移到RNA的自旋列（例如Nucleospin RNA二）从马谢雷-内格尔和执行列DNase的消化和洗涤步骤（遵循制造商的协议）;洗脱RNA在25μLH 2 O </li><li>对于DNA纯化Qiagen公司的的DNeasy血液和组织工具的优化协议。平衡裂解物与等量缓冲AL和100％的乙醇，自旋式离心机的负载（10,000 RTĞ1分钟），并丢弃流过。 </li><li>加入500μL，其中包括5μLRNA酶（1毫克/毫升）AW1缓冲列，并在室温下孵育10分钟。自旋（1分，在10,000 g，RT），并丢弃流过。 </li><li>用500μl缓冲AW2洗柱，流过旋干空列（1分钟）15000克丢弃。 </li><li>加入预热（70℃）缓冲AE和10分钟的孵育列在70°C。离心洗脱（1分钟万克），重新申请流过重复离心步骤。 </li></ol>使用分光光度计测定DNA和RNA的浓度。一个典型的PVN内冲产量〜600毫微克DNA和〜400毫微克的RNA。定量PCR（qPCR）的基因表达分析，我们通常使用〜100毫微克的RNA逆转录反应。 4。重亚硫酸盐转化亚硫酸氢钠是用来转换uracils非甲基化的胞嘧啶。相比之下，甲基化的胞嘧啶转换保护。因此，在最后的亚硫酸氢钠PCR的扩增测序检测到的所有胞嘧啶代表甲基化的胞嘧啶。 重亚硫酸盐转化导致大量的DNA降解，这可以是一个限制因素，在PCR分析。优化的反应条件下，最大限度地提高胞嘧啶转换，减少DNA片段，并保持单链DNA，即使在较低温度下可以实现使用EpiTect硫酸氢钠QIAGEN的试剂盒。在我们的手中〜200纳克从micropunches纯化的DNA提供足够量的亚硫酸氢钠反应起始材料。 为了防止在转换反应效率的差异，模板DNA的量应保持恒定在实验过程中处理所有样品。此外，序列读审议通过考察非转换非的CPGs率应为转换效率。这个比率应该超过98％。较低的值显示不完整或低效的重亚硫酸盐转化和底层的序列应该从分析中排除。 5。亚硫酸氢钠聚合酶链反应<ol><li>设计亚硫酸氢钠测序引物是特定的亚硫酸氢盐转化的DNA（见讨论）;甲基Primer Express软件（ <a href="https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&amp;catID=602121" target="_blank">https://products.appliedbiosystems.com/ab</a>/ EN /美国/ adirect / AB？CMD = catNavigate2及CATID = 602121），将有助于这一点，中试实验，以确定最佳退火温度。 </li><li> PCR主混合（一反应）准备如下： 2.5μL10x PCR缓冲液 0.5μL10 mM的dNTPs浓度 1μL10μM的正向引物 1μL10μM的反向引物 0.125μLQiagen公司的热启动Taq酶加 与H 2 O补至23μL </li><li>加入2μL亚硫酸氢钠处理DNA反应。 </li><li>放大使用下列条件： 1个周期6分钟95℃ 45-50周期1分钟95℃，最佳退火温度1分钟，1分钟72°C间 1个周期5分钟72℃ </li></ol> 7μlPCR产物的琼脂糖凝胶电泳分析，以验证扩增的大小和净化随后结扎剩余使用市售的PCR清洁试剂盒（PCR反应如马谢雷娜凝胶Nucleospin提取）。的情况下获得附加意外的PCR产物，凝胶净化建议。 6。亚硫酸氢钠测序高分辨率的DNA甲基化概况，从单一的克隆读数推导出可以检测DNA甲基化的微小变化，并确定监管区域，是顺应治疗（环境规划）。亚硫酸氢钠测序过程包括三个连续的工作步骤。首先，由之前的亚硫酸氢钠PCR获得的PCR产物连接到一个向量和细菌转化。其次，从单一的克隆PCR殖民地，以确定正确的插入大小。第三，积极菌落PCR的清理和遭受大染料的测序反应。经过清理的一步，产品电泳毛细管序。 6.1结扎和转型 注：我们经常使用的pGEM-T载体CLoning试剂盒（Promega公司）。在我们的经验，克隆效率的关键取决于上结扎插入。应测试不同的载体，重组克隆的低的情况下，多次获得。 <ol><li>建立连接反应： 5μL2X结扎缓冲区 1μLpGEM-T载体 1μl的T4连接酶 3μL清理PCR产物</li><li>通过吸取和孵育过夜混合反应在4°C。 注：结扎术可能也可以在室温下进行，以及为1小时。到晚上结扎重组克隆数增加，在4°C是首选。 </li><li>清理乙醇沉淀结扎产品： <ol class="lalpha"><li>加入1μL糖原，1μL3M NaAc和25μL100％的乙醇，在液氮中1分钟的连接反应和沉淀。 </li><li>离心机（15000克30分钟，在4°C间），弃上清。 </li><li>用300μL清洗70％的乙醇和离心机（15,000克20分钟，在4°C间），弃上清和沉淀干燥，在室温下（10分钟）。 </li><li>重悬浮颗粒，在10μL的H 2 O </li></ol></li></ol> 6.2结扎产品electrocompetent细菌转化<ol><li>预冷电试管（1毫米宽）冰和冰解冻等份electrocompetentDH5α，细菌。 </li><li>加入45μLDH5α，细菌10μL清理结扎产品（见上文），并转移到试管。 </li><li>改造1.5千伏，200Ω，15μF的细菌，并添加1毫升预热后直接脉冲传递哽咽介质。 </li><li>在37°C，1小时恢复细菌传播LB /氨苄青霉素与经IPTG / X-Gal的涂层板100μL悬浮。 </li><li>孵育板一夜之间在37°C。 </li></ol> 6.3菌落PCR 注：殖民地PCR技术从单一的克隆进行，以确保插入规模的预测。延迟的颜色从蓝/白筛选，结扎，低聚引物对纳入或截断的PCR产物中的意外重组事件的发展，否则可能导致故障的测序结果。我们经常使用的T7和SP6引物扩增克隆插入额外约150 bp的载体序列，这种方法的结果。的T7引物用于测序反应在后面的步骤。 <ol><li>建立了殖民地，在96孔板PCR反应。对于一个反应使用： 3微升2.5毫米MgCl 2的 2.5μL10倍的Taq缓冲 1.5μL10毫米的dNTP 2μL2.5毫米的T7引物 2μL2.5毫米SP6的底漆 Fermentas公司的Taq聚合酶1μL 填补与H 2 O 25μL </li><li>免除/主组合的96孔板每孔25μL。 </li><li>挑板枪头和浸正（白）克隆到PCR反应。 </li><li>扩大使用FOL必须符合以下条件： 1个周期4分钟95°C间 ℃，30日在第56号第10次30秒94°C和30秒72°C间 ℃，30日在第48号第30次30秒94°C和30秒72°C间 1个周期5分钟，72°C间</li><li>装载5μL菌落PCR和琼脂糖凝胶确定包含正确的插入大小的反应。 </li><li>含有所需扩增的殖民地PCR的清理使用一个市售套件（Machery内格尔Nucleofast）的。 </li></ol> 6.4大染料终止反应和测序<ol><li>准备大染料反应法师组合。对于一个反应使用： 1μLH 2 O 0.5μL大染料试剂 1.5μL测序缓冲</li><li>免除微升/ 3以及每一个96孔板，每以及加入2μl和清理菌落PCR产物，用下列参数运行一个热循环反应： 10秒35的周期在96℃，5秒，在50℃，4分钟在60°C </li><li>大染料的反应是使用商业试剂盒（Millipore公司蒙太奇96测序清洁套件）清理和处理上的毛细管测序仪（如ABI的3100的DNA）。 </li><li>序列分析，BIQ分析器（ <a href="http://biq-analyzer.bioinf.mpi-sb.mpg.de/" target="_blank">http://biq-analyzer.bioinf.mpi-sb.mpg.de/</a> ）的在线工具BISMA（ <a href="http://biochem.jacobs-university.de/BDPC/BISMA/" target="_blank">http://biochem.jacobs-university.de/BDPC/BISMA/</a> ）得出的甲基化模式的研究DNA区域（ 图4）。 </li></ol> 7。代表结果获得洞察ELS的AVP的表达和甲基化状态的影响，C57BL/6N小鼠，根据上述工作流程处理。简言之，一组C57BL/6N小鼠为Subjected给ELS，而对照组离开不受干扰。室旁核和视上核（SON），拳打脚踢同时分离DNA和RNA的单拳。 RNA的反转录和，AVP成绩单qPCR分析和归HPRT和GAPDH看家基因表达水平。 DNA是亚硫酸氢钠处理，与AVP增强（ 图4a）和PCR产物的特异性引物扩增，克隆和测序。至少有20个从每个鼠标/ PCR克隆进行分析，以确定甲基化PCR扩增（ 图4b）中的CPGs频率。与对照组相比，ELS的诱导CpG10，CpG12，CpG13 AVP增强Cpg14（P &lt;0.05，N = 6-8动物）1显着的甲基化，暗示后生本监管区域标记通过早期的生活经验。相比之下PVN内，甲基化AVP增强在儿子说明表观遗传标记（ 图4c）的组织特异性的ELS的影响。 DNA甲基化状态分析CpG10和AVP控制动物的基因表达（N = 6）负相关指向的DNA甲基化在AVP的基因表达（ 图4d）微调的作用可见一斑。 <img alt="图1" src="/files/ftp_upload/3938/3938fig1.jpg" /> 图1。的 Micropunches得到控制和生命早期强调小鼠大脑解剖。继DNA和RNA的分离，是由QRT-PCR基因表达，而亚硫酸氢钠处理的DNA扩增和克隆纯化产品是在一个合适的载体，允许重组克隆鉴定蓝色/白色选择。正确插入尺寸验证菌落PCR前开展大染料的反应和处理毛细管序。甲基化模式是通过适当的软件工具，可视化。 <a href="http://www.jove.com/files/ftp_upload/3938/3938fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3938/3938fig2.jpg" /> 图2。时间轴从DNA / RNA提取硫酸氢钠测序。 <img alt="图3" src="/files/ftp_upload/3938/3938fig3.jpg" /> 图3。同时提取DNA和RNA的工作流程。下丘脑室旁核，一个微小的AVP基因表达的大脑区域，打了一拳，被放置在400μL多伦多缓冲区，振荡，直到破坏和进一步匀浆通过注射器（29G）。匀浆，然后分裂RNA和DNA的纯化。拆分可以是1:1或不同比例在特定的实验问题。的匀浆应在几个小时内处理。 <img alt="图4" src="/files/ftp_upload/3938/3938fig4.jpg" /> 图4。在AVP轨迹在6周龄控制和ELS的动物CpG甲基化。 （一）AVP和催产素基因导向尾对尾的计划和区域间（IGR）分隔。外显子描绘开放（编号）框。的CpG残留的分布情况和各自的PCR扩增含CpG 10日至CpG14的大小和位置是一个大胆的行标记。 （二）在控制和ELS的动物PVN内AVP增强甲基化组（n = 6-8）。 （三）在控制和ELS动物的儿子（N = 8-10）甲基化的AVP增强。 （四）AVP基因的表达呈正相关与甲基在CpG10（N = 6）。 <a href="http://www.jove.com/files/ftp_upload/3938/3938fig4large.jpg" target="_blank">点击这里查看大图</a> 。

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没有利益冲突的声明。

后生编程日益被视为潜在的健康和疾病的一个重要机制。在这里，我们提出了一个同时隔离，从micropunches和后续步骤获得通过亚硫酸氢盐测序的DNA甲基化谱的DNA和RNA的精制方法。优化工作流程，允许在应对环境刺激大脑中的后生编程方便分析。此外，这种做法有利于功能的DNA甲基化和基因表达之间的关系，因为二者都是从同一样品分析的调查。最后，实验所需的动物数量可显着减少。 应遵循一定的预防措施和指引，而提出的工作流程执行。考虑非凡的复杂性和异质性脑和甲基化和表达模式，因为在细胞和组织的具体方式可能会有所不同，它是最重要的是micropunching在一个标准化的方式进行。作为一个点的情况下，AVP后生编程在paravaventriuclar检测，但没有在视上核（ 图4B-D），两个地区的下丘脑4 AVP表达。在这方面，来自同一组织冲床DNA和RNA的可用性是有利的RNA可以在某些情况下使用证明，正确的区域被打了个一定的组织特异性标记基因的表达。 ，虽然micropunching可以减少细胞的异质性，它必须记住，这种方法不会导致单细胞类型或种群的隔离。可能被视为额外的纯化步骤，以解决这一主题。 对于亚硫酸氢盐测序的PCR扩增以下的亚硫酸氢钠治疗的最佳引物设计是必不可少的。长度超过400 bp的PCR产物可以由B的问题，由于DNA降解isulfite治疗。还应指出，巢式PCR可以给偏见的结果，如果只有少数完整的模板有助于放大过程。设计应当是具体的修改扩增引物对，它们的DNA序列不应该含有CpG二核苷酸，以避免偏向甲基化扩增。另外，引物应该包含非CpG基胞嘧啶，以防止未修改或不完全转换的DNA扩增。进行热启动PCR扩增一些模板可能会受益。在任何情况下，对引物的适用性，应在试点实验验证，有价值的实验标本，以防止腐败。 亚硫酸氢钠测序代表一个站点特定的甲基化分析的标准方法，因为它是能够可靠和准确的检测甲基化的CpG单残留在等位基因特异的方式8。 PCR产物直接测序，不建议作为混合甲基Ø发CPG渣会出现一个C /双峰在电泳，它可以防止定量甲基化有关的网站。出于这个原因，中间克隆步骤进行，一些克隆测序（20-30），以确定甲基化的程度。焦磷酸测序的一种替代方法，以量化DNA甲基化。它还采用重亚硫酸盐转化和亚硫酸氢钠PCR而是克隆和测序的扩增受到直接的焦磷酸测序反应，其中一个CpG残留的甲基化状态是作为一个C / T单核苷酸多态性，可以很容易量化阅读。这两种方法检测9甲基化水平相似，但自克隆步可以省略焦磷酸测序技术是更多的时间效率。然而，根据模板只有40-100个核苷酸测序一次，这样几轮不同的测序引物与焦磷酸测序是必要的。这是一个关键的限制，给予THADNA T更高的金额（约1微克每反应）是必需的，可预见的超过金额可从微小的脑组织拳。因此，在地区范围内的几个碱基的初始扫描，由焦磷酸测序似乎不太适合作为单一的克隆读数。然而，对小区域的利益焦磷酸测序鉴定后应予以考虑。 总的来说，上述协议提供了一个后生变化的脑micropunches快速和成本效益分析，准确，高效和简化的方法。多个标本，可以在一个单一的运行和处理的方法是合适的运行时从中间大小的实验样本。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DNeasy血与组织套件</td><td> Qiagen公司</td><td> 69506 </td></tr><tr><td> nucleospin的RNA II </td><td>马歇雷的内格尔</td><td> 740955.50 </td></tr><tr><td> Epitect硫酸氢钠试剂盒</td><td> Qiagen公司</td><td> 59104 </td></tr><tr><td> nucleospin提取</td><td>马歇雷的内格尔</td><td> 740609.50 </td></tr><tr><td> pGEM-T载体克隆试剂盒</td><td> Promega公司</td><td> A3600 </td></tr><tr><td> nucleofast 96 </td><td>马歇雷的内格尔</td><td> 743100.50 </td></tr><tr><td>蒙太奇96测序清洁大行动</td><td> Millipore公司</td><td> LSK09624 </td></tr><tr><td> hotstar的Taq聚合酶</td><td> Qiagen公司</td><td> 203203 </td></tr><tr><td> Taq聚合酶</td><td> Fermentas公司</td><td> EP0401 </td></tr></tbody></table>

optimized analysis of dna methylation and gene expression from small, anatomically-defined areas of the brain

暴露于饮食，药物和早期生活逆境中，在生活的1,2敏感窗口可以导致持久的基因表达的变化，有助于生理和行为表型显示。这样的环境规划是可能增加易感性代谢，心血管疾病和精神疾病3,4。 DNA甲基化和组蛋白修饰的基因与环境对话的调解被认为是在关键工序，也出现下衬的环境规划5。在哺乳动物中，DNA甲基化通常包括甲基胞嘧啶5位内的CpG二核苷酸的背景下，除了共价。 CpG甲基化发生在一个高度的组织和细胞特异性的方式使其成为一个挑战，研究，脑细胞的异质性高，组织数量有限的离散小区域。此外，Because基因表达和甲基化紧密相连的活动，增加值，可以得到比较这两个参数在同一样品。 在这里，一步一步的协议，为大脑中的后生编程调查（ 图1）提出使用“母婴分离”说明目的的早期生活逆境的典范。协议描述的DNA和RNA可以同时分离出的大脑差异中年鼠标micropunches的准备，从而在同一样品中的DNA甲基化和基因表达分析。

Watch this Scientific Journal Video about Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain at JoVE.com

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

一个精简的工作流程，研究DNA甲基化和基因表达变化后早期生活压力。从新生鼠和离散脑组织隔离母体分离开始，我们代表的协议，同时为随后的亚硫酸氢钠测序和RT-PCR分析DNA和RNA分离脑组织拳。

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 ...

optimized-analysis-dna-methylation-gene-expression-from-small

Research

JoVE Journal

Neuroscience

18.7K 次观看.  Max Planck Institute of Psychiatry. 一个精简的工作流程，研究DNA甲基化和基因表达变化后早期生活压力。从新生鼠和离散脑组织隔离母体分离开始，我们代表的协议，同时为随后的亚硫酸氢钠测序和RT-PCR分析DNA和RNA分离脑组织拳。

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

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

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

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 ...

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 ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

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

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

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 ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

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 ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...