我们要感谢CIBIO（特伦托大学）和中国北车研究院神经科学的行政人员寻求帮助。乔瓦尼·普罗文扎诺是由韦罗内西基金会（米兰，意大利）博士后奖学金支持。这一工作是由意大利教育部大学和研究（2008年PRIN补助＃200894SYW2_002和打2010至2011年批＃2010N8PBAA_002到YB），特伦托大学（CIBIO创业补助金，以YB）和马拉松式节目基金会（拨款＃GGP13034到YB）。

所有实验均符合欧盟指令六十三分之二千〇一十/ EU进行，并批准了卫生意大利外交部。 1，动物保健，住房和治疗<ol><li>执行使用小鼠按照各自的机构动物护理准则的所有实验方案。 </li><li>维持动物在12小时光照/黑暗周期的食物和水可随意获得。 </li><li>众议院在小鼠2-4组，在标准尺寸的聚碳酸酯鼠标笼锯末垫料每周更换一次。 </li><li>使用年龄和性别匹配的小鼠，因为对疾病的易感性可以与年龄和性别的神经发育障碍的不同的鼠标型号而异。 </li><li>选择动物的适当数量达到统计学显着性（至少10只小鼠基因型的行为研究和4只小鼠基因型的免疫组织化学实验的）。 </li></ol> 2. Morris水迷宫（MWM） <ol><li>使用一个圆形罐（直径为100厘米，高度为50厘米）。 <ol><li>放置一些视觉提示对周围的坦克的房间隔断。 </li><li>放置一个直径10厘米的平台到罐在四个假想象限之一的中心的固定位置。保持在同一象限的所有培训课程的所有审判隐藏的平台。 </li><li>填用自来水池，直到平台为1cm的水面上方。平衡水的温度应接近22℃;这个步骤可能需要几个小时。加入热水，以缩短平衡水温度所需要的时间。 </li><li>添加大约1.5-2升白，无毒涂料，使水不透明。 </li></ol></li><li>轨道征收程序<ol><li>设置MWM的跟踪系统与标准的CCD摄像头和视频板。选择一个视频跟踪系统，以监视和分析几个变量，如路径长度，ESCAPE延迟和时间花费在每个象限中。 </li><li>划分舞台四个象限，调整检测设置，以确保动物和背景之间最佳的对比度进行现场跟踪。 </li><li>指定平台区域。 </li><li>设置最大的试验时间为60秒。 </li></ol></li><li> MWM测试<ol><li>前行为测试，允许小鼠在20-25分钟的适应期在一个地区，他们不能看到游泳池或空间线索。 </li><li> （一天连续两次审判，它们之间20-30分钟间隔）训练每只小鼠9天。 </li><li>切换计算机和摄像机并开始播放视频跟踪系统软件。 </li><li>就拿老鼠的尾巴，将其放入水中，用它的头指向了游泳池边，从四个起始位置（北，南，东，西）中的一个开始。对于每个小鼠，开始位置应该是不同的，并且在各试验pseudorandomized。 </li><li>让鼠标游泳，搜索platfORM最多60秒。一旦动物达到平台，允许它停留在平台上，持续20秒。 </li><li>如果鼠标没能找到时间内平台上，轻轻引导鼠标的平台，通过它的尾巴抱着它，让它呆在那里，持续20秒。一旦动物已完成全部两项试验，用毛巾擦干其关闭，并把它放回笼子。 </li><li>训练期间中的每一天，重复相同的过程（步骤2.3.4-2.3.6）。 </li><li>最后一次训练后的步道上进行9天的探索实验。 </li><li>从池中取出平台。 </li><li>让鼠标游泳和搜索平台，最多60秒。 </li><li>定期检查水温度，以便它应该是相同的（22℃）和清理出池。 </li></ol></li></ol> 3.准备从灌流动物的节<ol><li>牺牲小鼠在MWM的末端。每四个基因型的小鼠之前必须要麻醉euthaniz根据当地和国际准则通货膨胀。 </li><li> transcardially灌注动物（切右auriclewith剪刀，以允许出血和引入一个蝴蝶套管进入左心室灌注）用磷酸缓冲盐水（PBS）为4-5分钟，随后在PBS中的4％多聚甲醛为10-15分22。 </li><li>解剖和后固定在4％多聚甲醛的大脑在4℃下至少12小时。 </li><li>直到所需的切片放置在含有在4℃在0.1M PBS中的15ml 0.02％的叠氮化钠和存储50毫升聚苯乙烯锥形管的大脑。 </li><li>剪切和vibratome切割之前删除的小脑。 </li><li>胶大脑直立与切割部位，用强力胶，到vibratome的固定板。 </li><li>切脑入20-40微米厚的冠状切片，在串行顺序整个背海马，适当的vibratome。 </li><li>收集vibratome片，用画笔和转移到24多孔板包含用于每个井1毫升的0.1M PBS中。 </li><li> （组织可能被用于最多一年）店铺部分为短期的PBS或长期在0.02％叠氮化钠的PBS中，在4ºC。 </li></ol> 4.免疫组化<ol><li>转印3-5背海马切片每只小鼠，于24多孔板含有500微升的0.1M PBS中每个孔中。 </li><li>用500微升的0.1M PBS（5分钟，3次，用温和振荡）洗涤包含在每个部分好。 </li><li>淬灭内源过氧化物酶活性，通过将切片在1％的H 2 O 2的PBS 20分钟，轻轻摇动。 </li><li>在0.1M PBS中，轻轻摇动（3×5分钟）洗涤切片。 </li><li>组织的通透性:在PBS中孵育切片5分钟在0.2％的Triton X-100。 </li><li>在上述步骤过程（4.4; 4.5），使足够封闭缓冲液（10％山羊血清和0.1％的Triton-X100的PBS）在整个实验（numbe段 - [R×100微升×3步）。 </li><li>注意:从在其中二次抗体（缀合到生物素）中的由相同物种使用血清。 </li><li>防止非特异性抗体结合，通过将切片在封闭缓冲液（100μl/孔部）1小时，在室温（RT）下温和搅拌。 </li><li>孵育稀释在封闭缓冲液中过夜，在4℃，温和搅拌在初级抗体（的pERK）部分。 </li><li>在0.1M PBS中，轻轻摇动（3×5分钟）洗涤切片。 </li><li> （如在步骤4.6中使用相同）处理1小时，在室温下缓慢搅拌稀释在封闭缓冲液:孵育缀合有生物素（250 1）的次级抗体。 </li><li>制备ABC试剂同时，因为抗生物素蛋白和生物素至少需要30分钟，在室温下，以复合物。 </li><li>准备根据制造商的协议ABC试剂的所需体积。 </li><li>在0.1M PBS中，轻轻摇动（3×5分钟）洗涤切片。 </li><li> IncubatË部分在室温与ABC试剂45分钟。 </li><li>在0.1M PBS中，轻轻摇动（3×5分钟）洗涤切片。 </li><li>准备工作DAB解决方案，涉及的每一步DAB必须做通风柜作为DAB为致癌和致畸。 </li><li> DAB传输解决方案，以一个新的板块由塑料移液管。 </li><li>在含DAB手工工作溶液慢慢旋流板的板放置节，以允许承受的最大部分。 </li><li>监测显微镜DAB反应，直到有足够的信号开发（2-5分钟）。 </li><li>停止在所希望的颜色强度的反应洗涤在0.1M的PBS（3×5分钟）该组织。 </li><li>使用漂白剂停用通风柜任何剩余的DAB底物溶液中过夜，并根据实验室规定进行处置。 </li><li>明胶山段浮在PBS涂幻灯片。然后干燥它们在室温下至少3-4小时。 </li><li>在50％的脱水段顺序地，70％，95％和100％的乙醇，每次2分钟，并明确在100％二甲苯5分钟。 </li><li>盖玻片用封固剂和在通风橱离开干燥过夜。 </li></ol> 5.细胞计数<ol><li>算的pERK阳性细胞，三到五节，在背侧海马的水平应每只动物进行分析。 </li><li>从各部分中，在放大200倍的使用适当的显微镜获取多个亮场图像，然后使用图像处理软件将它们组装。 </li><li>关于使用ImageJ的免费软件TIFF转换拼接图像进行细胞计数。 </li><li>计数结束二点五十八计数为100×100微米的每个箱子的pERK染色的细胞在门区及颗粒细胞层。 </li><li>细胞密度将表示为每计数窗口（100×100微米）标记的细胞的数目。 </li><li>执行统计分析，以评估基因型之间显著差异。这可以用学生t测试或ANOVA，然后通过使用商业软件适当事后检验。在P &lt;0.05设置统计显着性水平。 </li></ol>

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作者什么都没有透露

在这里，我们提供了一个的pERK免疫协议为揭示神经元激活在EN2以下MWM - / -小鼠，神经发育障碍的小鼠模型。被检测EN2的CA3区振作水平降低- / -突变体相比，WT。不同于在CA3区，pERK的阳性神经元的普遍增加什么观察物代替在两个门区检测和EN2的GCL - / -小鼠与WT相比。在ERK磷酸化水平这一趋势相反的一种可能的解释可能依赖的pERK感应以下MWM子的具体机制。 <p class="jove_content"...

神经发育障碍包括多种异构疾病如唐氏综合征，脆性X染色体综合征（FXS），Rett综合征，神经纤维瘤病，结节性硬化症和ASD，其中中枢神经系统（CNS）的发育和成熟期间早期干扰的基胎儿期1。这些发育的大脑功能障碍可引起运动功能，语言，学习和记忆过程中深刻的，终身的影响。遗传和环境因素的过多，在过去的几年里2,3-已牵涉在神经发育障碍的发病机制。即使临床表型的分子机制仍然是未知的，上面提到的研究结果已允许的这些疾病的几个小鼠模型的发展。学习和记忆的赤字已经确定了一些，这些小鼠模型，如+/- TSC1，TSC2的+/-，NF1 +/-和EN2 - / -小鼠2,4-7。在神经发育障碍的领域的一个重要的挑战是底层记忆和学习功能障碍细胞和分子过程的识别。学习或记忆时激活所选的信号通路可诱导特异性基因的转录，并最终导致从头蛋白质的合成。立即早期基因（即早基因）活化和蛋白依赖性突触修饰迅速诱导的脑的神经元响应于神经元活动和行为训练8,9。 在涉及的信号通路神经纤维瘤蛋白赤字已与神经发育障碍学习障碍有关。神经纤维瘤是NF1基因的产物，其突变导致神经纤维瘤病1型，一个复杂的遗传综合征的特点是神经系统的肿瘤，行为和电机的延迟，和认知迪萨bilities 10。小鼠杂合NF1缺失局限于抑制性神经元表现出的长时程增强的早期阶段（LTP），以及在MWM 5,11,12损害空间学习障碍。有趣的是，NF1缺陷在该小鼠模型导致过度活化在抑制性Ras信号在学习期间，导致增加ERK磷酸化，最后在GABA的释放，从这些神经元5的异常强化。 基于这些发现，神经元活动的行为任务之后的可视化表示的方式来重建涉及神经发育疾病的具体电路。这里所描述的免疫组化协议旨在评估和量化的ASD小鼠模型的认知缺陷以下MWM海马ERK的磷酸化水平。 MWM被广泛用来研究在啮齿类动物13,14海马依赖性的空间学习和记忆</s向上&gt;。我们决定使用ERK磷酸化作为任务依赖性海马学习的分子读出，由于ERK显示出具有在学习和记忆形成15了至关重要的作用。此外，ERK途径是必要的经验依赖性可塑性在显影视觉皮层16。最后，缺乏两个ERK亚型（ERK2）在CNS节目之一的小鼠标记在认知，情绪和社会行为17的异常，这表明ERK信号可能在神经发育障碍，如房间隔缺损的发病中起关键作用。 我们使用ENGRAILED 2淘汰赛（EN2 - / - ）小鼠神经发育障碍的典范。 EN2 - / -小鼠显示出解剖和行为"ASD样"特征，包括前脑interneurons 18的损失，ASD相关基因19表达降低，降低的社交性，和受损的认知灵活性6,7,20。空间学外贸纳克和存储器的缺陷，如那些在MWM检测，尤其稳健在EN2 - / -小鼠6,7-和可能相关于在ASD患者21中观察到的认知障碍。此外，我们发现，空间学习MWM中受损与神经纤维瘤缩小表达有关，增加的pERK水平EN2的门区- / -成年小鼠7。在这里，我们提出详细的协议振作这个ASD小鼠模型以下MWM的免疫组化特征。

<table><tbody><tr><td>EthoVision XT 8</td><td>Noldus Information Technology</td><td></td><td>This software platform is not a requirement &amp;ndash; there are many other behavioral softwares on the market.</td></tr><tr><td>Tempera Paint</td><td>Giotto - Fila Group Company</td><td></td><td>White and liquid, non toxic. Used to prepare opaque water in the Morris water maze.</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1200</td><td>Equivalent models from other companies can be used.</td></tr><tr><td>24 well plate</td><td>Sigma</td><td>CLS3524</td><td></td></tr><tr><td>100% ethanol</td><td>Fisher Scientific</td><td>A406-20</td><td>Used to make ethanol gradient for dehydration prior to slide mounting.</td></tr><tr><td>Xylene</td><td>VWR</td><td>66004-950</td><td>Toxic - to be used under hood. Change xylene every month depending on use.&amp;nbsp;</td></tr><tr><td>Sodium Azide</td><td>Sigma</td><td>&amp;nbsp;S2002</td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>P3813-10PAK</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Sigma&amp;nbsp;</td><td>T-8787</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Sigma</td><td>H1009-100ML</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Abcam</td><td>G9023-10ML</td><td></td></tr><tr><td>ABC kit Vectastain&amp;nbsp;</td><td>Vector Laboratories</td><td>PK-6100</td><td>Add in a volume of 5 ml of PBS 2 drops of reagent A, mix and then add 2 drops of reagent B and mix.</td></tr><tr><td>DAB peroxidase substrate</td><td>Vector Laboratories</td><td>SK-4100</td><td>Add in a volume of 5 ml ddH&lt;sub&gt;2&lt;/sub&gt;O: 2 drops of buffer stock solution and mix; 4 drops of DAB and mix; 2 drops of H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; and mix.</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>pERK antibody</td><td>Cell Signaling Technologies&amp;nbsp;</td><td>4370</td><td>Dilution 1:500</td></tr><tr><td>Biotinylated Goat Anti-Rabbit IgG Antibody</td><td>Vector Laboratories</td><td>BA-1000&amp;nbsp;</td><td>Dilution 1:250</td></tr><tr><td>SuperFrost Slides&amp;nbsp;</td><td>Carl Roth</td><td>1879</td><td></td></tr><tr><td>Coverslips</td><td>Fisher</td><td>12-548-B</td><td></td></tr><tr><td>DPX</td><td>Sigma</td><td>317616</td><td>Mounting medium for slides. Equivalent mounting medium can be used.</td></tr><tr><td>Microscope</td><td>&amp;nbsp;Zeiss&amp;nbsp;</td><td>Axio Imager.M2</td><td>Equivalent microscope can be used.</td></tr><tr><td>Adobe Photoshop</td><td>Adobe Systems, San Jose, CA</td><td></td><td>To assemble images.</td></tr><tr><td>ImageJ Software</td><td>National Institute of Health</td><td></td><td>Free software can be downloaded at http://rsb.info.nih.gov/ij/</td></tr><tr><td>SigmaPlot 11.0</td><td>Systat Software Inc. (USA)</td><td></td><td>Equivalent softwares for statistical analysis can be used.</td></tr><tr><td>Prism 6</td><td>GraphPad Software, Inc. (La Jolla, CA, USA)</td><td></td><td>Equivalent softwares for statistical analysis can be used.</td></tr></tbody></table>

immunohistochemical visualization of hippocampal neuron activity after spatial learning in a mouse model of neurodevelopmental disorders

感应磷酸化细胞外调节激酶（的pERK）是学习相关神​​经元激活的可靠的分子读出。这里，我们描述一个的pERK免疫协议来研究海马神经元激活的轮廓在暴露于一个空间学习任务的特征在于神经起源的认知缺陷小鼠模型。具体来说，我们使用的pERK免疫研究神经元激活以下Morris水迷宫（MWM，经典的海马依赖学习任务）中ENGRAILED-2基因敲除（EN2 - / - ）小鼠，自闭症谱系障碍模型（ASD）。相比于野生型（WT）对照，EN2 - / -小鼠显示出在MWM显著空间学习障碍。 / - - MWM后，在EN2的特定海马子场中检测到在的pERK阳性神经元的数目显著差异小鼠相比，WT动物。因此，我们的协议可以稳健地检测在不同在ASD的小鼠模型相关联的海马依赖性学习障碍的pERK阳性神经元。更一般地，我们的协议可以应用于调查其特征在于认知缺陷都遗传或药理学小鼠模型的海马神经元激活的轮廓。

这里所描述的协议被设计来可视化，通过免疫组织化学，海马神经元活动的MWM之后的特定标志物在神经发育障碍的小鼠模型中的表达。所有本文中所示的实验数据取自我们最近的工作7。男性和女性WT和EN2 - / -年龄匹配的同窝成人（3-5个月;重量= 25-35克）用杂交配获得的使用。二十二个小鼠（11％基因型）被用于MWM。 8只（4％基因型）处死在MWM探针试验结束并用于免疫组织化学实验?...

Watch this Scientific Journal Video about Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders at JoVE.com

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

我们描述了一个协议，免疫组织化学研究海马神经元激活的轮廓暴露于空间学习任务为特征的神经发育起源的认知缺陷小鼠模型。此协议可应用到特征的认知缺陷都遗传或药理学小鼠模型。

海马神经元活动的可视化免疫组化空间后学习神经发育障碍的小鼠模型

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

immunohistochemical-visualization-hippocampal-neuron-activity-after

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.2K 次观看.  University of Trento. 我们描述了一个协议，免疫组织化学研究海马神经元激活的轮廓暴露于空间学习任务为特征的神经发育起源的认知缺陷小鼠模型。此协议可应用到特征的认知缺陷都遗传或药理学小鼠模型。 

视频: Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

科研

行为学

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

神经科学

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

体内 小鼠海马体中小胶质细胞的慢性双光子成像

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

将人干细胞衍生的GABA能神经元移植到出生后早期小鼠海马体中以减轻神经发育障碍

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using ...

Imaging Hippocampal Neuronal Activation In Mouse Brain Sections

<a href='https://appjove.unicartagenaproxy.elogim.com/v/52919/immunohistochemical-visualization-hippocampal-neuron-activity-after'>Source: Provenzano, G., et al.&#160;Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders. J. Vis. Exp.&#160;(2015)</a>This video demonstrates immunohistochemical imaging to visualize hippocampal neuronal activation. Stained brain sections reveal darker regions under a bright-field microscope, indicating activated neurons.

Source: Provenzano, G., et al.&#160;Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of ...

JoVE Encyclopedia of Experiments

Neuroimaging

Structural & Functional Brain Imaging

Fluorescence Immunostaining of Rat Hippocampal Neurons within a Microfluidic Chip

Source: Nagendran, T., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/58421">Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons.</a> J. Vis. Exp. (2018)
This video demonstrates immunofluorescence staining of rat hippocampal neurons cultured within a microfluidic chip to analyze neuronal maturation and connectivity.

微流控芯片内大鼠海马神经元的荧光免疫染色

1. Fluorescence Immunostaining within the Chip

	Prepare 4% formaldehyde fixation solution in PBS (4% formaldehyde, 1 &#181;M MgCl2, 0.1 &#181;m CaCl2, ...

Neuropathology

Trauma

Immunofluorescence Staining for Visualizing Proliferated Neural Stem Cells in Mouse Brain Sections

Source: Quintanilla, L. J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59237">Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for staining newly proliferated neural stem cells in brain tissue sections using EdU incorporation, followed by immunofluorescence to visualize specific proteins, enhancing the understanding of cell dynamics and proliferation.

免疫荧光染色用于观察小鼠脑切片中增殖的神经干细胞

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunohistochemistry

	...

Developmental and Pediatric Disorders

Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence

<a href='https://appjove.unicartagenaproxy.elogim.com/v/56153/evaluation-of-synapse-density-in-hippocampal-rodent-brain-slices'>Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).</a>This video demonstrates a method for visualizing synaptic markers in hippocampal brain slices using confocal immunofluorescence analysis. The slices are immunostained with primary antibodies targeting presynaptic and postsynaptic markers, followed by fluorophore-conjugated secondary antibodies. The labeled markers are then visualized using confocal microscopy to evaluate the synaptic architecture through their colocalization.

Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).This video demonstrates a method for ...

Synaptic & Cellular Imaging

An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis

Source: Ansorg, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52551">Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis.</a> J. Vis. Exp. (2015)
The video illustrates an immunofluorescence staining method used to examine neurogenesis in the adult hippocampus. Initially, a mouse brain section undergoes treatment with primary antibodies directed at marker proteins associated with neurogenesis in neuronal progenitor cells. Next, it is labeled with secondary antibodies tagged with fluorophores. The brain section is then observed under a confocal microscope to distinguish various subtypes of progenitor cells, based on the expression patterns of distinct combinations of marker proteins.

一种检测成人海马神经发生的免疫荧光染色技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tissue ...

海马神经元活动的可视化免疫组化空间后学习神经发育障碍的小鼠模型

摘要

探索更多视频

此视频中的章节

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

树突分支中的海马区对小鼠的齿状回评

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

体内小鼠海马体中小胶质细胞的慢性双光子成像

将人干细胞衍生的GABA能神经元移植到出生后早期小鼠海马体中以减轻神经发育障碍

Imaging Hippocampal Neuronal Activation In Mouse Brain Sections

微流控芯片内大鼠海马神经元的荧光免疫染色

免疫荧光染色用于观察小鼠脑切片中增殖的神经干细胞

Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence

一种检测成人海马神经发生的免疫荧光染色技术

海马神经元活动的可视化免疫组化空间后学习神经发育障碍的小鼠模型

摘要

探索更多视频

此视频中的章节

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

树突分支中的海马区对小鼠的齿状回评

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

体内 小鼠海马体中小胶质细胞的慢性双光子成像

将人干细胞衍生的GABA能神经元移植到出生后早期小鼠海马体中以减轻神经发育障碍

Imaging Hippocampal Neuronal Activation In Mouse Brain Sections

微流控芯片内大鼠海马神经元的荧光免疫染色

免疫荧光染色用于观察小鼠脑切片中增殖的神经干细胞

Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence

一种检测成人海马神经发生的免疫荧光染色技术

体内小鼠海马体中小胶质细胞的慢性双光子成像