This work has been funded by the Israel Science Foundation Grant (ISF # 393/12), Israel Centers of Research Excellence Grant (I-CORE 1796/12), German-Israel Foundation Grant (GIF # 2299-2291.1/2011) and the Marie Curie Career Integration Grant (FP7-PEOPLE-2013-CIG #618201). Initial steps in the project were funded by an AXA postdoctoral fellowship to AC. We acknowledge the generous startup funds provided by the Edmond and Lily Safra Center for Brain Sciences.
Critical reading by members of the Citri lab is greatly appreciated.

注:该协议遵循耶路撒冷希伯来大学的动物护理指引。 1，准备学联解决方案<ol><li>如表1中所述制备ACSF溶液。车型1升在DDH 2 O（&gt; 18兆欧的纯度），使渗透压〜300毫渗透摩尔/升以适当添加水或氯化钠。 </li></ol> 2，设备及室内设置该设备用于监控的可卡因诱导的运动行为由行为室（50×45cm）上连接有内光源，风扇和相机（或红外传感器），并配有在其内的有机玻璃箱（30×30厘米）该小鼠可以自由移动，同时其运动受到监视。在行为测试应1小时之间灯之后进行到1小时灯之前关闭，在常规的光/暗循环。动物要坚持在每天同一时间进行培训。 <ol><li&gt;连接​​摄像机或红外线系统到一个适当的系统监测和记录小鼠的自发行为。 </li><li>清洗每次试验用小鼠设施和/或除臭剂提供消毒后的腔室，以防止嗅觉提示偏压，并确保适当的消毒。 </li></ol>重要提示:当处理的小鼠安静，冷静和谨慎。 3，动物准备<ol><li>众议院5 C57BL6雄性小鼠（6-12周龄〜25〜35克的重量）每笼，在一个配有12小时的光/暗周期和免费获得食物和水，按照标准和当地的动物的要求护理委员会的指导和协议。在启动实验，权衡小鼠和日志数据。可卡因是根据小鼠的体重后施用。 </li><li>在开始第一次庭审前将全部含有老鼠进入室内行为30分钟的笼子里。 </li><li> ，观察者所有的在实验中使用的小鼠对实验者处理剂，注射剂，并在行为设置监测。这是通过加入250μl盐水连续3次每日腹膜内注射，如下所述来实现。 </li><li>辖腹膜内注射250μl的生理盐水。紧接在注射，将鼠标在一个行为室来监测运动活性15分钟。每天重复这个动作，连续3天，在相同的时间。 </li><li>在第四天，分化的小鼠分成两组。制备可卡因的原液在2毫克/毫升的盐水中。管理一个单一的注射可卡因溶液（最后20毫克/千克）的实验组根据鼠标的重量（ 如 25 G滑鼠-注入250微升，而30克鼠标将获得300微升）。施用生理盐水的对照组。紧接射出，将鼠标20分钟的行为室监测自发活动（炎热的典型盟友，前15分钟进行监控）。 </li><li>可卡因注射后，牺牲小鼠在不同时间点（0，1，2，4，8和24小时以下的注射）。重要的是，确保参考老鼠（0时刻）将不会收到任何注射的这一天。由于其重要性，就必须有参​​照组的复制。 </li></ol> 4，解剖的伏隔核<ol><li>麻醉的小鼠用异氟烷在可卡因/盐水注射后适当的时间。异氟醚必须直接排放走出了房间。因此，该程序应在通风橱中进行。 </li><li>通过测试后足部反射监测麻醉深度。捏爪子引发的动物提款回应。动物，显示了反射是不是麻醉的手术水平，而不是在国家安乐死。 </li><li>提取的大脑: <ol><li>断头安乐死鼠标。 </li> &lt;LI&gt;删除上面的头骨中间的皮，切用锋利的剪刀头骨。 </li><li>将剪刀在那里的脊髓进入脑中（枕骨大孔）点，使两侧切口。 </li><li>从同一点，使沿矢状缝长矢状切。在到达嗅球，夹在左侧和右侧。 </li><li>有镊子，小心地取出颅骨，抓颅骨各占一半牢固，拉在一旁，小心不要按到或损伤大脑。 </li><li>取出大脑，而通过使用一个倒置的微勺铲切断脑神经。 </li></ol></li><li>将大脑中的冰冷ACSF溶液1-2分钟来冷却和硬化。 </li><li>从学联取出大脑，灯芯多余的液体用纸巾，创造小脑和大脑的其余部分之间的冠状切口和胶水的脑，有喙的部分朝上，上vibratome阶段。 </li><li>切割的伏隔核: <ol&gt; <li>维持学联的解决方案，在冰冷的气温在vibratome的切片室。在200微米到伏隔核（NAc的）部分被清楚地识别根据Paxinos和Franklin小鼠脑图谱（约1.8毫米前到前囟门;注意胼胝体，前连合的位置和形状的形状和心室，以及在脑切片的整体的形态）。在这一点上，创建两个400微米的部分，从该伏隔将解剖。 </li><li>根据立体镜，用细刀片从解剖切片的伏隔核区域了。根据Paxinos和Franklin小鼠脑图谱的伏隔核的定义，并可以方便地通过在组织上的刀片4快速传递解剖中相应的部分。 （ 图2）。 </li><li>将立刻伏隔段在离心管和存储900微升的Trizol裂解试剂在-80°C，直到RNA的抽行动。 </li></ol></li></ol> 5，RNA提取和cDNA反转录<ol><li>解冻含核管伏在Trizol试剂裂解试剂部分在室温下和均化用1ml注射器连接至25g针溶解物直至组织完全均化（至少15-20倍）。第一几轮是困难的，并且需要推压管的壁的组织，以将其分解到小块可能进入针的尖端。 </li><li>以确保均质，吸管溶解物到QIAshredder微型旋转柱和离心机以12,000 xg离心1分钟。 </li><li>根据制造商的说明吸管溶解物到新的离心管中，并提取RNA。存储所述RNA在-80℃直到使用。 </li><li>测量RNA浓度，使用生物分析仪或等同设备分析的完整性和质量。使用100-500纳克的RNA每一轮的cDNA的制备有Random六聚体引物。 </li></ol> 6，引物设计和检测引物<ol><li>选择良好的引物:为获得最佳引物对mRNA转录的定量PCR，请按照下列要求（ 图3）: <ol><li>设计引物含有不超过17-28个碱基。避免核苷酸重复，因为他们推动错配。 </li><li>设计引物之间56-68℃（最佳60-64℃）熔化温度（Tm值）。该引物对中的Tm值应该是在彼此的1℃。 </li><li>请注意，该产品的尺寸最好应在80和150个基点之间。 </li><li>确保引物中的至少一个包括一个外显子 - 外显子交界处，或扩增子中含有大量的内含子（内含子跨越），以避免基因组DNA污染的扩增。 </li><li>为了提高引物的设计采用基于引物3（对于实施例参照表2）的可靠的软件。 </li></ol></li><li>测试PRimers:为了确保引物的专一性和效率，控制定量PCR反应应进行: <ol><li>用积极的样品（含cDNA模板为所有相关的引物对）和滴定的基因（ 如 8从〜10毫微克的初始浓度3倍稀释）一式两份平行反应。包括无模板对照，这对于用于反应的溶液中的污染控制。 </li><li> （ - 1 /斜率）-1 10（）}×100，使得它们的最佳斜率的曲线是- 3.333（ 即 10（ - 1 / 3.333）= 2）（ 图4）:使用下面的公式计算出的引物效率。 </li><li>确定扩增的特异性。特异性扩增证明由共同的扩增产物所有的熔解曲线的单一主峰，而脱靶扩增会出现一个明显的偏差FROM单大峰。 </li></ol></li></ol> 7，定量PCR分析<ol><li>前置放大: <ol><li>综合定量PCR分析是基于限制的RNA的量与大量的引物对的并行查询。因此，预扩增步骤是必不可少的。在此步骤中，cDNA经受预扩增14-18个循环的所有将被用于查询该样品在未来（最多800个引物对）中的引物的混合物。 </li><li>稀释于TE（低EDTA）的所有引物，以50为针对特定目标扩增（STA）。 </li><li>池一起所有的50μM引物的1微升等分设置要包括在该STA反应，高达800测定。 </li><li>制备预混物和样品的STA，如表3所述。通过制备混合为需要被放大的样本数的110％允许误差。 </li><li>等分3.75微升STA预混每个样品。添加1.25微升的cDNA的每一个。 </li><li>涡流以混合反应并离心10秒。 </li><li>将STA的反应在热循环和周期，在表4所示。 </li></ol></li><li>核酸外切酶I（ExoI）治疗: <ol><li>稀释的外切余如表5所示。 </li><li>加入2微升稀释ExoI（4 U /μL），以每5微升的STA反应，漩涡，降速（&gt; 6000 XG），并将其放置在热循环仪，37 ℃下进行30分钟，然后在80℃下进行15分钟。 </li><li>稀释的最终产品，以适当的浓度进行测试。用TE缓冲液（加43μLTE缓冲液到7微升TSA样品）。 </li><li>存储稀释STA产品在-20°C或立即使用。 </li></ol></li><li>底漆和样品安装在定量PCR: <ol><li>制备的样品，如表6。根据选择的实验中，芯片准备混合，并添加样品适当。 </li><li>敏捷射孔样品混合解决方案，最少20秒，而离心机（&gt; 6000 XG）至少30秒。制备的反应可以被存储为短的持续时间，在4℃，直至该芯片已准备好装载。 </li><li>如表7中所述制备的测定。 </li><li>搅动测定混合物最少20秒，离心机（&gt; 6000 XG）的至少30秒降速所有组件。 重要提示:涡彻底离心吸取之前的所有样品制备和分析解决方案集成到芯片的入口。如果不这样做，可能会导致数据质量的下降。 注意:每个引物的终浓度为5μM的在入口和500nM的最终反应。 </li></ol></li><li>底漆和加载动态数组国际金融公司（集成流体通路）芯片。在IFC芯片具有入口的样品和测定法，以及指定的入口（累加器）为用于加压的微流体腔室的控制管道流体（矿物油）（Figu重新4A）。 <ol><li>注入控制线流体到芯片上的每个累加器。 </li><li>移除并从芯片的底部丢弃蓝色保护膜。 </li><li>将芯片插入相应的国际金融公司（IFC）控制器（控制器MX的48.48动态阵列国际金融公司（IFC）和国际金融公司（IFC）控制器HX的96.96动态阵列国际金融公司），然后运行首相（113X）脚本的48.48动态阵列国际金融公司（IFC）和总理（136X）脚本的96.96动态阵列国际金融公司（IFC），以素片。 </li><li>当剧本完成后，请从国际金融公司控制器底漆芯片。 </li><li>移液管将5μl各测定混合物和5μl各样品混合到其入口。 </li><li>返回芯片到IFC控制器。 </li><li>利用国际金融公司控制器软件，运行负载混合（113X）脚本的48.48动态阵列国际金融公司）或混合装载（136X）脚本的96.96动态阵列国际金融公司装载样品和检测到芯片中。当负载混合脚本已经完成，除去负荷版芯片从国​​际金融公司控制器。 </li><li>装载在热循环仪的芯片，创建一个新的芯片运行（根据制造商的指导方针），包括熔解曲线分析。使用热循环仪软件，确保该反应室被正确识别，并允许该反应进行至完成。 </li></ol></li></ol> 8，数据分析<ol><li>质量保证:为了确保数据的质量，通过寻址稀释度曲线验证引物的质量（如上述）。通过观察扩增子的熔化曲线监测扩增的特异性，其中峰值应最佳地紧密排列29。 </li><li>可视化:对于大批量的高通量定量PCR获得的数据可视化，使用软件生成的热图，其中表现为颜色编码的强度。此外，显示单个基因作为内衬散点图的转录动力学。 </li><li>出版:出版基因表达的结果，建议遵循MIQE准则发布实时定量PCR实验，详细说明了必须报告的定量PCR实验，以确保其相关性，准确性，正确的解释，和可重复性的最少信息。 </li></ol>

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We have nothing to disclose.

从脑组织以下行为范式成功的表征基因的表达依赖于:行为范式中1）认真小鼠的处理; 2）快速，精确的利益组织的解剖; 3）RNA的保护措施以确保RNA的完整性; 4）引物和实验布局精心规划，以及精度和对细节的关注，准备的qPCR分析。 描述的步骤的目的是表征诱发一种行为体验的转录活动动态;因此，认真注意是必要的，以确保该行为范式所经历的小鼠确实表示打算由研究者的经验。例如，即使在这样的简单范例作为行为敏可卡因，鼠标的经验可以受许多干扰因素:鼠标在归属笼之前，当然先有经验periment;转移到试验室的方法;在试验室内的光线，声音和气味曝光;处理被实验者的经验;疼痛/不适引起的IP注射;暴露于一种新的实验，并随访至安乐死的点指定的行为的经验。这些经历每一个可能，孤立，促进诱导小鼠不同脑区的转录程序。因此，必须小心，以确保转录程序测量所需的范例，并且不附带现象与范例相关联。在可卡因经验的情况下，这是很容易通过仔细的实验​​和对细节的关注，以及简单的控制实验，其中所有的参数都保持不变，但是媒介物（盐水）注射到小鼠而非可卡因实现。根据我们的经验，盐水注射引起小规模的转录程序相比，可卡因曝光，这表明该范例压倒性地使所关心的现象的调查。 悉心照顾，应采取限制的鼠标经验的范围，而它仍处于活动状态，因此有必要对小鼠的冷静和谨慎处理。与此相反，以下麻醉，快速和精确的工作是必不可少的，由于在RNA中的代表性的快速变化（在分钟的时间刻度）和RNA的固有不稳定性的可能性，这可能会迅速降解，一旦细胞遭到破坏，从而可能破坏所致的经验转录轮廓。因此重要的是从颅骨迅速取出大脑，在激冷环境（1-2分钟），迅速解剖伏隔在冰冷条件。一旦大脑被冷冻，用于转录变化或RNA降解的可能性是显著减少，但只有当有关节放在Trizol试剂和冻结，我S中的组织的完整性和转录信息的表示确保。 在这方面，值得一提的是立即早期基因表达的转录动力学发生在快速时间尺度，经常达到峰​​值之前的1小时以下的刺激。在所述的实验范例的上下文中，转录动力学的询问被限制的小时刻度，以降低样品之间的变异性。为次短于1小时，在定时的微小变化可能导致大的变化的观察转录变化的幅度。因此，在1小时的时间点已经被选择用于分析，因为该时间点是实验上更容易与精确度来执行，并且不容易受到变化时，在实验几分钟差的水平。 传统上，组织micropunch已被许多实验室的组织样本的解剖。有有很多的理由喜欢组织的手工显微​​切割。冲头通常是由不锈钢制成，并且是不透明的。因此，定位打孔的精确和一致的位置可能会非常棘手。此外，冲头不会允许，如果有在初始位置确定一个错误的任何修正，以制成。最后，从冲床中提取的组织有时可以证明棘手，而且有潜在的失去组织或结转样本之间。使用细手术刀将组织的显微切割使实验者更精确的控制，并提供关于样品的纯度安全。 在RNA提取和直至合成cDNA，工作环境需要保持无RNA酶的污染物的来源，并且工作应使用无RNA酶的工具，一次性物品和试剂来进行。在这个阶段，最小的污染，很容易损坏的辛苦赚来的RNA样品。保持样品íCE冷是用于最小化，可以修改该转录读出，以及尽量减少可能影响样品的完整性酶活性神经活动是至关重要的。额外的药理抑制剂有时加入，以减少神经兴奋性，但通常这些都不是必不可少的。 继逆转录成cDNA，重点转向确保从成绩单的综合分析得到的结果的有效性。第一步是确保适当的定量PCR引物利用，为此，我们强烈建议测试底漆效率和特异性上定义的RNA源的稀释曲线。最佳的PCR引物为目标的特定和有效扩增。特异性扩增，表明扩增仅与靶序列的，显而易见的，在熔解曲线分析的单个离散的峰，而有效扩增，表明在每个周期中，与靶序列的数量是复制，具有效率接近100％。由于微流控芯片的qPCR的高通量特性，这些实验需要仔细规划，以确保列入了显著数阳性和阴性对照的。优选的是，包括在每一个芯片的运行相一致的控制，使得从对照和实验条件的结果的直接比较。在实验过程中的设置，这是至关重要的不交叉污染的孔中。由于引错以及少量会引起不必要的对象的放大，要特别注意吸取引物时服用。 用于从大脑大多数组织样品是困难的是肯定的夹层中只有感兴趣的组织中提取的，没有污染不相关的大脑区域，这可能会混淆的结果的分析。为此目的，重要的是控制探测基因预计其表达被排除在感兴趣的组织。确定基因表达的空间分布格局有用的工具是艾伦脑图谱（ <a href="http://www.brain-map.org/">http://www.brain-map.org/</a> ）。 在数据分析的深入讨论超出了本手册的范围，但应该指出的是认真尝试，值得认真分析。确保了一些相关的参考基因被包括在每一轮的qPCR的是很重要的。这些基因将作为用于标准化，以确保相似数量的RNA被检测。归一化，执行第一到参考基因的样品中，然后与参考老鼠的实验（"时间0"），在应用"ΔΔCT方法"。 在这个手稿中描述的协议的目的是研究转录动力学以下小鼠中的伏隔核可卡因经验。然而，这是很容易ADAPTEd表示的小鼠的其它任何经验的研究，只要适当的控制实现的，而经验的定时被很好地定义。很显然，这个协议也可以用于转录动力学在组织中的神经系统以外的研究，以及实施。 但是应当强调的是，使用微流体为基础的qPCR全面转录谱是仿形的转录事件的具有成本效益的方法，但取决于所关注的靶基因的先验知识。这些都通过无偏见纹的方法，如微阵列和深度测序通常获得。因此，在一个大项目的工作流程，全面的定量PCR分析可以提供大量的数据，但优先应遵循系统的前公正的表征。 还值得指出的是，本文描述的用于获得组织样本的过程中，也可以对研究实施荷兰国际集团的各种其他细胞活动 - 蛋白质组学和蛋白质修饰，代谢组学，脂类组学和表观遗传标记。

大脑的充满活力的组织能够认知和行为的灵活性。经验是通过神经元之间的连接的结构和强度的变型在大脑3编码。这个"经验依赖性可塑性"是诱导特异性基因表达，可提供必要的蛋白质的突触结构和强度4的变形例的模式的结果。基因调控网络的识别中介长期记忆的形成是分子神经科学的核心原则，并期望识别转录程序的主导因素将提供洞察调节记忆的形成，以及对目标的基本原则治疗神经变性和神经精神障碍。转录程序展开在时间上定义的波，其中每一个编码的基因不同的字符，这是当d重要ifferent阶段的信号事件1,2的结果的执行情况。因此重要的是要解决的转录动力学进行了详细的时间时间表，以便确定诱导的基因的全面补充，并洞察他们的潜在功能，根据自己归纳的动态。 吸毒是造成药物滥用的神经回路在大脑5,6长期影响的经验依赖可塑性强大的表单。最初，急性暴露于药物可能导致成瘾的发展和过渡到长期使用。上下文信息是网瘾发展的关键因素。药物相关环境线索在药物滥用者的头脑中分配显著重要性。上下文信息，提醒过往用药经验滥用药物可诱发复吸毒品的渴求，甚至禁欲以下长时间的药物暴露7,8。因此，重要的临床挑战成瘾-成瘾的倾向，停药后症状已经消退9复发甚至长。 行为敏可卡因是可卡因经验的毒瘾机制的研究很有用一个简单的模型。在本作中持久过敏引起的慢性接触到滥用药物被广泛研究的模式，鼠类首先习惯于注射生理食盐水（腹腔，IP）的一个新的环境中（一个开放的领域腔中的自发活动监测） ;然后，他们收到可卡因每天注射，在旷场箱，而它们的活性被监测10（ 图1）。这种行为模式通常导致的自发行为（8-12倍以上的基线活动）11，这是保持以下停止注射可卡因数月的强劲敏，显示了PERV的形成的用药经验asive记忆痕迹。 奖励的神经回路，自然参与加强行为的一个物种的成功（ 例如喂食，性别）必不可少的，是由药物滥用，加强药物相关的行为12,13的利用。由药物滥用的经验增强的分子和细胞机制似乎是类似的底层声明或语义记忆在其他大脑结构14的形成机制。因此，该行为敏化模型的稳健性，使其成为有吸引力的模型系统来研究的经验依赖性可塑性机制。 伏隔核（NAC）是大脑奖励环路的中央集成商，并已被广泛使用成瘾5,6的发展有关。瘾的形成取决于在伏隔核的新的蛋白质的转录，并在鲁棒的结构清晰的转录程序duction是在伏隔核以下可卡因经验15-19 观察到可卡因曝光的急性转录反应是可能的，多层次的，以适应于强诱导刺激，并直接生产新的蛋白质的功能即负责通过照射诱发的药物6,19-22的结构和电学的改变。 为了促进的经验依赖性可塑性在大脑中的分子机制的研究中，一个协议被描述为转录动力学的脑组织样品中的下列行为操作的全面分析。该协议说明的行为经验研究的生活更轻松实验室环境 - 行为敏可卡因，利用微流体动态数组进行转录分析。中描述的协议显然不限于学习吨他细胞核中行为敏化的背景下伏，而是可以应用到大量的行为范例和大脑区域。事实上，这个协议可以被应用到身体组织中的外脑，和各种经历或生物体的操作的影响。 该协议被粗略地分为四个步骤。在第一步骤中，将动物进行的行为模式;在第二步骤中，组织是显微切割的;在第三步骤 - 的mRNA的纯化，逆转录和探测，并在最后的步骤中的数据进行了分析。 在研究转录动力学的情况下，精确定时的经验和定义可能是最重要的实验参数来控制。出于这个原因，我们的行为选择的模式是，行为敏可卡因，一种系统，其允许实验者控制的高含量超过experien的参数CE。提供额外的行为范式，使精确定时和解决不同型号的经验依赖可塑性和记忆的形成。这些模型包括恐惧条件23，急性丰富环境24,25，新物体探索26项黑暗饲养27的视觉体验。然而，行为敏可卡因是一贯稳健的操纵行为，创造一个高度普遍的记忆痕迹而持续数月以下的可卡因经验28。 大脑切片，随后是伏隔核的手工显微​​切割。一直以来，我们的经验，从快速制备脑切片手工显微切割提供提取相关的行为范例组织的最可靠和快速的方法，并且有经验的，组织的边界变得明显和容易地识别。另外，精细的切片可以prepar编，接着通过激光捕获显微切割。虽然这种方法能够高度限定的感兴趣区域的分界，它是慢（因而冒不稳定的mRNA的丢失），繁琐，并且需要昂贵的专用设备（配有激光捕获设置的显微镜）。本文所定义的协议，也可以适用于单细胞转录分析，通过使用膜片移液管29在视觉上识别细胞的细胞质中的手动抽吸。要注意的是所描述的协议提供了人口平均，而这是极可能的是，在大多数情况下，在组织内的细胞中，只有亚群实际参与反应的经验是很重要的。这是感兴趣的，从特定的细胞群响应经验内轮廓的转录以选择性方式，但这些方法的讨论已超出当前范围。 对于mRNA的纯化，反转录和定量PCR的查询中，组织是通过细针，其次是市售试剂盒的使用传递给它打乱了（有关详细信息，请参阅表8）。的选择是通过用这些方法，这确保了高品质的RNA和从下游应用健壮的结果可靠提取经验得知。 而该协议描述了用于高通量定量PCR使用动态阵列，样品可以用终点PCR，低通量定量PCR，基因表达的微阵列或深度测序被探测的基因表达。偏爱高通量定量PCR使用动态阵列是由于mRNA的脑细胞得到下列行为范例是常常限制量的事实。动态数组提供了一个平台，使成绩单高效综合分析从一个单一的实验大量平行样品。最初的收购微流体系统（通常是机构PU后rchase），实验是相对便宜的运行。根据这一分析，可以用更昂贵的平台，以寻找新的成绩单（由微阵列或RNAseq）与动态数组提供质量保证全面的参考来进行样品的进一步查询。最后，对数据进行分析，标准的方法被利用​​。对于可能出现的问题的具体指针将在协议的文本进行讨论。 该协议是最适合感兴趣的是他们的利益制度进行彻底的调查调查，研究多个条件和重复。该协议也是谁已经磨练中（通过微阵列或RNAseq实验）在50-500基因的兴趣，他们有兴趣在反复询问一个子集，研究者最合适的。

<table><tbody><tr><td>Virusol</td><td>Oriek Medical</td><td>J29D</td><td></td></tr><tr><td>Isoflurane, USP 100%</td><td>MINRAD INC</td><td>NDC 60307-110-25</td><td></td></tr><tr><td>RNeasy plus Universal Mini Kit</td><td>QIAGEN</td><td>73404</td><td></td></tr><tr><td>QIAshredder</td><td>QIAGEN</td><td>79654</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription kit</td><td>Invitrogen</td><td>AB-4368814</td><td></td></tr><tr><td>TE Buffer</td><td>Invitrogen</td><td>1355656</td><td></td></tr><tr><td>Behavior Chamber (MDF; 50 x 45 cm)</td><td></td><td></td><td>Self assembled</td></tr><tr><td>Inner Perspex box (30 x 30 cm)</td><td></td><td></td><td>Self assembled</td></tr><tr><td>Camera and video recorder</td><td>Campden Inst.</td><td>CMD-80051</td><td></td></tr><tr><td>Media Recorder software</td><td>Noldus</td><td>NDS-NMR3-00M</td><td></td></tr><tr><td>Iris Scissors</td><td>FST</td><td>FST-14062-09</td><td></td></tr><tr><td>Vibratome</td><td>Campden Inst.</td><td>7000SMZ-2</td><td></td></tr><tr><td>Bioanalyzer</td><td>Agilent Technologies</td><td>The Agilent 2100 Bioanalyzer</td><td></td></tr><tr><td>Thermal cycler</td><td>Bio-Rad</td><td>1852048</td><td></td></tr><tr><td>Inverted microspoon spatula</td><td>Bochem Instrument GmbH</td><td>3213</td><td></td></tr><tr><td>Biomark HD Reader</td><td>Fluidigm</td><td>BMHD-BMKHD</td><td></td></tr><tr><td>Dynamic array Chip for 96.96 gene expression</td><td>Fluidigm</td><td>BMK-M-96.96</td><td></td></tr></tbody></table>

comprehensive analysis of transcription dynamics from brain samples following behavioral experience

在大脑和长期记忆巩固经验的编码依赖于基因转录。识别编码经验的特定基因的功能的分子神经科学的主要目标之一。此外，确定基因与特定行为的功能联系对于理解神经精神疾病的基础意义。感应的鲁棒转录方案已在下列各种行为操作小鼠的大脑中观察到。而一些遗传元件被用于循环地以下不同行为的操作和在不同脑核，转录方案是整体独特的诱导刺激物和它们所研究1,2的结构。 在本出版物，协议是针对操纵行为的描述，从小鼠脑核强大和全面的转录谱。该协议中演示了在细胞核中基因表达的动力学分析的情况下伏隔急性可卡因经验。随后到体内的经验定义，目标神经组织被解剖;其次是RNA纯化，反转录和利用微阵列为多个目标基因的综合定量PCR分析。该协议是着眼于全面的分析（处理50-500个基因）限制原料的数量，如小的大脑标本，甚至单个细胞。 该协议是对多个样品的平行分析（ 如单细胞，动态分析下列药物，病毒或行为紊乱）最有利的。然而，该协议也可以事先对全基因组研究中起到了样品的特性和质量保证通过微阵列或RNAseq，以及从全基因组研究中获得的数据的验证。

通过施加这个协议中得到的结果的质量在很大程度上取决于于许多参数。适当的试验计划将造成最小干扰的实验小鼠，如所测试的经验（在本例中，即暴露于可卡因）将在其最近历史上最占优势的经验，因此，将导致一个强大的和特异性的转录程序。 图1描述了实验计划行为敏可卡因，限定的时间点以下可卡因经验在其中的伏隔核是从小鼠解剖和分析。下一个关键点是，限定了精确的边界进行分析的适当组织的切除， 图2描述了在冠状面和矢状切片伏隔核的边界。优选地，所述夹层应该执行，使得薄NAc的组织的余量被排除的区域德恩的分析。这将确保一致的分析仅南汽，减少了可变性和来自包括周围组织产生的伪影的潜力。 当扩增限制性的RNA的量，如在小脑核的剥离，所必需的合适的检测信号进行放大的多个周期。因此，在任何定量PCR实验的关键点上，与少量的起始材料的工作时，进一步放大，是用于扩增的引物的特征， 图3描述了在确定最佳的引物的主要特点，和图4显示的引物的扩增曲线定向抗c-Fos和FOSB蛋白，这表明这些引物对扩增的靶转录物在每个周期〜100％的效率。以下引物验证和建立正确的实验范式，使转录从目标做卷烟明确评估UE，综合分析可以在Fluidigm公司的BioMark平台上执行，从而实现到9216平行的qPCR反应（在96.96格式; 图5）。这种高通量平台允许多个实验重复的平行分析，使用相同的实验条件下在单个芯片上。对于四倍重复实验数据，解决了转录诱导的c-Fos和FOSB蛋白急性可卡因的经验，都证明了在图6中。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51642/51642fig1highres.jpg" width="500" /> 图1:实验范式进行动态转录分析如下可卡因的经历。 （一）实验方案-小鼠习惯于处理和注射3天，而在消音室的行为监控自发活动通过视频跟踪。小鼠然后受可卡因的经验;单次曝光=急性可卡因经验;连续5次注射被称为慢性暴露。单注以下≥16天可卡因禁欲被称为可卡因的挑战。转录动力学得到解决在以下可卡因经验（1，2，4，8，24小时），不同的实验时间点（B）情节鼠标轨迹的行为腔内以下3天盐水注射或盐水+ A 3天单急性暴露于可卡因。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51642/51642fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51642/51642fig2highres.jpg" width="500" /> 图中的伏隔核2，地点在小鼠大脑。厚厚的白线标出削减确定B的位置<html> （B）矢状切面，伏隔核（A）冠状切面的oundaries。 （图片改编自艾伦脑图谱参考图集图片: <a href="http://atlas.brain- map.org/atlas?atlas=1&amp;plate=100960352">HTTP://atlas.brain- map.org/atlas?atlas=1&amp;plate=100960352</a> ; <a href="http://atlas.brain- map.org/atlas?atlas=2&amp;plate=100883869">网址://atlas.brain- map.org/atlas?atlas=2&amp;plate = 100883869</a> ）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51642/51642fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51642/51642fig3highres.jpg" width="500" /> 图3。规划定量PCR引物 。准则规划定量PCR引物，对特异性，稳定性和兼容性的定义。<html> 2fig3highres.jpg"目标="_ blank将"&gt;请点击这里查看该图的放大版本。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51642/51642fig4highres.jpg" width="500" /> 图4。评估底漆效率。 Fos和FOSB蛋白的使用Fluidigm公司国际金融公司阵列扩增曲线（一）结果。使用来自小鼠伏隔核中提取和探查Fos和FOSB蛋白的表达的总RNA 7串行3倍稀释液产生标准曲线（B）的引物对为Fos和FOSB蛋白的效率是通过绘制循环阈值（评价克拉）在对倍稀释的样品的对数各稀释。引物的效率是从标准曲线的斜率计算出，如在文中所定义。稀释曲线上的扩增曲线和点是颜色匹配的。51642 / 51642fig4highres.jpg"目标="_ blank将"&gt;请点击这里查看该图的放大版本。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51642/51642fig5highres.jpg" width="500" /> 图5。综合定量PCR分析应用Fluidigm公司的平台。 （一）96.96动态阵列国际金融公司（IFC）的实时定量PCR检测。引物是装在位于右侧的入口，并且将样品在位于芯片的左边入口加载。这个数字已经被修改与Fluidigm公司实时定量PCR分析用户指南许可。（二）热图中的定量PCR结果，代表Ct值每口井的芯片。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51642/51642fig5highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <imgALT ="图6"FO:内容宽度="5英寸"SRC ="/文件/ ftp_upload / 51642 / 51642fig6highres.jpg"WIDTH ="500"/&gt; 图表达动态下的一种急性可卡因曝光6样品的结果 。 c-fos和FOSB蛋白的可卡因诱导表达显示为时间的函数。四实验重复，根据本文所定义的协议进行的平均值，在将显示，随着误差线描绘的平均值的标准误差。 <table border="0" cellpadding="0" cellspacing="0" width="336"><tbody><tr><td>试剂</td><td>兆瓦</td><td>毫</td><td> g / L的</td></tr><tr><td>氯化钠（Sigma-Aldrich公司，71376） </td><td> 58.44 </td><td> 119 </td><td> 6.95 </td></tr><tr><td> 碳酸氢钠 （Sigma-Aldrich公司，S6014） </td><td> 84.01 </td><td> 26 </td><td> 2.18 </td></tr><tr><td>葡萄糖（Sigma-Aldrich公司，G8270） </td><td> 180.16 </td><td> 10 </td><td> 1.8 </td></tr><tr><td>氯化钾（Sigma-Aldrich公司，P9541） </t<html> D&gt; <td> 74.55 </td><td> 2.5 </td><td> 0.186 </td></tr><tr><td>和NaH 2 PO 4（Sigma-Aldrich公司，S9638） </td><td> 137.99 </td><td> 1 </td><td> 0.138 </td></tr><tr><td> 硫酸镁⋅7H2 O（Sigma-Aldrich公司，M1880） </td><td> 246.5 </td><td> 4 </td><td> 0.99 </td></tr><tr><td> 氯化钙 （Sigma-Aldrich公司，C3011） </td><td> 147 </td><td> 4 </td><td> 0.59 </td></tr></tbody></table> 表1学联的解决方案。 <table border="0" cellpadding="0" cellspacing="0" width="508"><tbody><tr><td>软件名称</td><td>应用</td><td>网站位置</td></tr><tr><td>罗氏Probefin​​der </td><td>引物设计</td><td> http://qpcr.probefin​​der.com/organism.jsp </td></tr><tr><td> NCBI的引物爆炸</td><td>引物设计</td><td> http://www.ncbi.nlm.nih.gov/tool​​s/primer-blast/ </td></tr></tbody></table>ve_content"&gt; 表2软件列表。 <table border="0" cellpadding="0" cellspacing="0" width="429"><tbody><tr><td>试剂</td><td>体积/反应（微升） </td><td> 48反应10％超龄（微升）量</td><td> 96反应10％超龄（微升）量</td></tr><tr><td> 2X的TaqMan前置放大器主要混合液（Applied Biosystems公司） </td><td> 2.5 </td><td> 132 </td><td> 264 </td></tr><tr><td> 500牛米（10X）汇集混合底漆</td><td> 1 </td><td> 52.8 </td><td> 105.6 </td></tr><tr><td>水</td><td> 0.25 </td><td> 13.2 </td><td> 26.4 </td></tr><tr><td>基因</td><td> 1.25 </td><td></td><td></td></tr><tr><td>总成交量</td><td> 5 </td><td></td><td></td></tr></tbody></table> 表3 STA反应溶液。 <table border="0" cellpadding="0" cellspacing="0" width="426"><tbody><tr><td>条件</td><td>持有</td><td> 10-14周期</td><td>持有</td></tr><tr><td>温度</td><td> 95°C下</td><td> 95°C下</td><td> 60ºC </td><td> 4ºC </td></tr><tr><td>时间</td><td> 10分钟</td><td> 15秒</td><td> 4分钟</td><td> ∞ </td></tr></tbody></table> 表4热循环条件下进行预扩增。 <table border="0" cellpadding="0" cellspacing="0" width="509"><tbody><tr><td>组件</td><td>每5μL样品（微升） </td><td> 48个样品有超龄（微升） </td><td> 96个样品有超龄（微升） </td></tr><tr><td>水</td><td> 1.4 </td><td> 84 </td><td> 168 </td></tr><tr><td>核酸外切酶I反应缓冲液</td><td> 0.2 </td><td> 12 </td><td> 24 </td></tr><tr><td>核酸外切酶I当20单位/微升</td><td> 0.4 </td><td> 24 </td><td> 48 &lt;/ TD&gt; </tr><tr><td>总成交量</td><td> 2.0 </td><td> 120 </td><td> 240 </td></tr></tbody></table> 表5 ExoI反应溶液。 <table border="0" cellpadding="0" cellspacing="0" width="596"><tbody><tr><td>样品混合物</td><td>每个入口组分体积（微升） </td><td>每进气流量与超龄（微升） </td><td>为48.48动态阵列国际金融公司（微升）量（120个样本） </td><td>为96.96动态阵列国际金融公司（微升）量（120个样本） </td></tr><tr><td> 2X SsoFast EvaGreen Supermix低ROX（Bio-Rad公司，PN 172- 5211） </td><td> 2.5 </td><td> 3 </td><td> 180 </td><td> 360 </td></tr><tr><td> 20X的DNA结合染料上样试剂（Fluidig​​m公司，PN 100-3738）绿帽</td><td> 0.25 </td><td> 0.3 </td><td> 18 </td><td> 36 </td></tr><tr><td> STA和ExoI处理的样品</td><td> 2.25 </td><td> 2.7 </td><td></td><td></td> &lt;/ TR&gt; <tr><td>总</td><td> 5 </td><td> 6 </td><td></td><td></td></tr></tbody></table> 表6样品预混合的解决方案。 <table border="0" cellpadding="0" cellspacing="0" width="533"><tbody><tr><td>组件</td><td>每次进量（μl） </td><td>每进气流量与超龄（微升） </td><td>为48.48动态阵列国际金融公司（微升）量</td><td>为96.96动态阵列国际金融公司（微升）量</td></tr><tr><td> 2X含量装载试剂</td><td> 2.5 </td><td> 3 </td><td> 180 </td><td> 360 </td></tr><tr><td> 1X的DNA悬浮缓冲液（TE低EDTA） </td><td> 2 </td><td> 2.4 </td><td> 144 </td><td> 288 </td></tr><tr><td> 50为每一个混合正向和反向引物</td><td> 0.5 </td><td> 0.6 </td><td></td><td></td></tr><tr><td>总成交量</td><td> 5 </td><td> 6 </td><td></td><td></td></tr></tbody&gt; </table> 表7分析混合的解决方案。 <table border="0" cellpadding="0" cellspacing="0" width="389"><tbody><tr><td>该试剂/材料的名称</td><td>公司</td><td>目录编号</td></tr><tr><td> Virusol </td><td> Oriek医疗</td><td> J29D </td></tr><tr><td>异氟醚，USP 100％ </td><td> MINRAD INC。 </td><td> NDC 60307-110-25 </td></tr><tr><td>的RNeasy加上通用Mini试剂盒</td><td> QIAGENE </td><td> 73404 </td></tr><tr><td> QIAshredder </td><td> QIAGENE </td><td> 79654 </td></tr><tr><td>大容量cDNA逆转录试剂盒</td><td> Invitrogene </td><td> AB-4368814 </td></tr><tr><td> TE缓冲液</td><td> Invitrogene </td><td> 1355656 </td></tr></tbody></table> 表8中列出的化学品/试剂。 <table border="0" cellpadding="0" cellspacing="0" widtH ="389"&gt; <tbody><tr><td>设备名称</td><td>公司</td><td>目录编号</td></tr><tr><td>行为商会（中密度纤维板，50×45cm）上</td><td>自组装</td><td></td></tr><tr><td>内的有机玻璃箱（30×30厘米） </td><td>自组装</td><td></td></tr><tr><td>相机和录像机</td><td>卡姆登研究所</td><td> CMD-80051 </td></tr><tr><td>媒体录像机软件</td><td> Noldus </td><td> NDS-NMR3-00M </td></tr><tr><td>虹膜剪</td><td> FST </td><td> FST-14062-09 </td></tr><tr><td> Vibratome </td><td>卡姆登研究所。 </td><td> 7000SMZ-2 </td></tr><tr><td>生物分析仪</td><td>安捷伦科技公司</td><td>安捷伦2100生物分析仪</td></tr><tr><td>热循环仪</td><td> Bio-Rad公司</td><td> 1852048 </td></tr><tr><td>倒microspun锅铲</td><td> Bochem仪器有限公司</td><td> 3213 </td></tr><tr><td>的BioMark HD重阿德</td><td> Fluidig​​m公司</td><td> BMHD-BMKHD </td></tr><tr><td>动态阵列芯片为96.96基因表达</td><td> Fluidig​​m公司</td><td> BMK-M-96.96 </td></tr></tbody></table> 表9。设备清单。

Watch this Scientific Journal Video about Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience at JoVE.com

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

转录动力学的脑组织样本在行为经验综合分析

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

comprehensive-analysis-transcription-dynamics-from-brain-samples

Nanyang Technological University

Research

Education

JoVE Core

JoVE Journal

Introduction to Psychology

Behavior

Unit 1

Motivation and Emotion

SCIENTISTS IN ACTION

11.6K 次观看.  The Hebrew University of Jerusalem. This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays. 

视频: Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

科研

遗传学

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

单细胞的转录组学分析

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

神经科学

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

一个人脑组织中的染色质含量

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

生物学

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

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 ...

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 ...

<meta charset="utf8"></head><body>用于分析 MRI 环境中对外部线索的行为反应的管道

Profiling Maternal Behavior Responses During Whole-Brain Imaging

Recent advancements in whole-brain imaging tools have enabled neuroscientists to investigate how coordinated brain activity processes external cues, influencing internal state changes and eliciting behavioral responses. For example, functional magnetic resonance imaging (fMRI) is a noninvasive technique that allows for the measurement of whole-brain activity in awake, behaving mice using the blood oxygenation-level-dependent (BOLD) response. However, to fully understand BOLD responses evoked by external stimuli, it is crucial that experimenters also assess behavioral responses during scans. The MRI environment poses challenges to this goal, rendering commonly employed methods of behavioral monitoring incompatible. These challenges include (1) a restricted field of view and (2) the limited availability of equipment without ferromagnetic components. Presented here is a behavioral video analysis pipeline that overcomes these limitations by extracting valuable information from videos acquired within these environmental constraints, enabling the evaluation of behavior during the acquisition of whole-brain neural data. Employing methods such as optical flow estimation and dimensionality reduction, robust differences can be detected in behavioral responses to stimuli presented during fMRI scans. For example, representative results suggest that mouse pup vocalizations, but not pure tones, evoke significantly different behavioral responses in maternal versus virgin female mice. Moving forward, this behavioral analysis pipeline, initially tailored to overcome challenges in fMRI experiments, can be extended to various neural recording methods, providing versatile behavioral monitoring in constrained environments. The coordinated evaluation of behavioral and neural responses will offer a more comprehensive understanding of how the perception of stimuli leads to the coordination of complex behavioral outputs.

<meta charset="utf8"></head><body>在全脑成像过程中分析母体行为反应

Presented here is a video analysis pipeline that overcomes challenges in behavioral monitoring within MRI environments, allowing for the detection of uninstructed behavioral responses to external cues. This analysis will facilitate a more comprehensive understanding of evoked internal state changes and brain-wide activity.

在全脑成像过程中分析母体行为反应

Recent advancements in whole-brain imaging tools have enabled neuroscientists to investigate how coordinated brain activity processes external cues, ...

行为学

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

转录动力学的脑组织样本在行为经验综合分析

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转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

单细胞的转录组学分析

一个人脑组织中的染色质含量

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

在全脑成像过程中分析母体行为反应

在全脑成像过程中分析母体行为反应

在全脑成像过程中分析母体行为反应

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后