这项工作得到了巴塞尔大学生物医学系和瑞士国家科学基金会（31003A-116624）。

1。建立器官小脑切片培养出生后小脑切片文化准备从8 P（8）鼠标幼崽使用静态培养方法10。在我们的实验室中，我们使用B6CF1小鼠。在一些实验中也转基因小鼠，用于表达EGFP的浦肯野细胞的选择性。编制片文化需要大约30分钟，每鼠标的小狗，也就是3个小时的一窝6小鼠的幼崽。所有步骤都是在无菌条件下在层流工作台与消毒的外科手术器械进行。 <ol><li> P8鼠标幼仔小脑解剖从大脑，在冰冷却的制备培养基（MEM（Gibco公司产品编号11012）与glutamax 1:100，pH值7.3），使用在立体显微镜。 </li><li>小脑被切成350微米厚的片，在矢状方向使用一个McIlwain的组织菜刀。 </li><li>片被放置在微孔细胞培养插入（approx.6片每插入，Millipore公司的PICM03050），并培养在6孔板中，用0.75毫升每孔中的培养液（48.35毫升MEM，马血清，25毫升的Eagle培养基25毫升"，"1毫升glutamax朕一个无菌的10％葡萄糖，0.65毫升溶液，pH 7.3）与5％CO 2的潮湿气氛中，在37℃下所有的组织培养试剂，Invitrogen公司自Gibco公司。 </li><li>药物治疗通常开​​始在体外（DIV3）在3天中的所需浓度的药物加入到培养基中。佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）和（RS）-3,5 - 二羟苯甘氨酸（DHPG）从TOCRIS英国。由于许多药物需要使用DMSO或乙醇制备的原液（例如PMA）应小心向培养基中的总溶剂的添加量不超过1％（7.5微升每孔）。续药物一起的培养基每2 或第3天的变化。 </li></ol> 2。固定和免疫抑制泰宁的器官小脑切片培养小脑的一个重要优点是，抗体可用专门和明亮的小脑的主要的神经元​​，即浦肯野细胞和颗粒细胞染色。可以揭示出浦肯野细胞的树突状形态的，通过免疫染色用抗钙结合蛋白calbindin D28K。 <ol><li>对于固定培养，培养基中除去，并小心地加入每孔含有Millicell小插入与小脑切片附着在膜3毫升冷在0.1M磷酸盐缓冲液（pH7.4）中的4％多聚甲醛中。培养物固定过夜，然后除去固定液，培养物用磷酸缓冲液漂洗。 </li><li>对于免疫染色使用以下抗体:兔抗钙结合蛋白D-28K（Swant，贝林佐纳，瑞士）在1:1000用于选择性地可视化1:500的浦肯野细胞的单克隆抗-NEUN（Chemicon公司，Millipore公司），用于选择性地可视化制粒LE细胞12。兔抗GFP是从Abcam公司，英国。荧光二Alexa的抗体分子探针，Invitrogen公司。 </li><li>培养通过在0.1M磷酸盐缓冲液（封闭溶液）中加入3％正常山羊血清+ 0.3％TritonX100透性和阻止。反钙结合蛋白calbindin D28K和抗NEUN在封闭溶液和稀释的培养物培养含有抗体过夜，4℃下轻微的搅拌下，用封闭溶液。所有稀释液被表示为％（体积/体积）的Triton X100是取自10％的原液以便利移液。 </li><li>将培养用0.1M磷酸盐缓冲液中漂洗3倍，然后孵育适当的第二抗体，例如山羊抗兔Alexa的（568）和山羊抗小鼠Alexa的488 + 0.1％TritonX100 2小时，在0.1M磷酸盐缓冲液中1:500稀释室温。培养，然后用0.1M磷酸盐缓冲液漂洗3倍。 </li><li>将染色的切片从培养物中除去以及用画笔的Superfrost加载玻片安装在和与一个适当的安装介质，例如的Mowiol封片。现在可以被视为一个普通显微镜，荧光显微镜设备，或用激光共聚焦显微镜的文化。 </li></ol> 3。查看个人的浦肯野细胞在器官切片培养<ol><li>免疫组化与钙结合蛋白D28K后，浦肯野细胞的细胞质中明亮的染色，包括树突和轴突。由于浦肯野细胞的浦肯野细胞层致密取向，大多数的浦肯野细胞的树突状乔木，是重叠的，使得难以研究完整的心轴的一个单独的细胞。然而，由于在筹备过程中的一些浦肯野细胞的细胞死亡，在许多文化中一些地区存在浦肯野细胞密度降低。在这样的区域，它是能够找到与其他C不重叠的浦肯野细胞与一个完整的树突状乔木英语学习者。在这些细胞中，树突状乔木可以查看和分析到高尔基染色的质量相媲美。因为在小脑的浦肯野细胞的细胞是唯一的细胞类型表达钙结合蛋白D28K也被确定为浦肯野细胞。 </li><li>另一种方法是使用来自B6的培养; FVB-的Tg（PCP2-EGFP）146.244Yuza / J小鼠（这里称为PCP2-EGFP的小鼠，可从Jackson实验室）表达EGFP的浦肯野细胞的具体L7启动子11下。在切片文化生活的浦肯野细胞可以可视化表达EGFP。另外，也可以按照一个单一的细胞随着时间的推移的树突状形态。或者，固定后，EGFP表达的浦肯野细胞可以具有抗GFP抗体免疫染色。这两种方法都适用于染色器官文化的浦肯野细胞的树突，胞体和轴突。由于L7启动子在PCP2-EGFP小鼠每浦肯野细胞中不表达，而且是v表达小鼠（Kapfhammer，数据未示出）之间的浦肯野细胞的频率中切片ariations来自这些小鼠的培养使其更易于可视化和测量具有与其他的浦肯野细胞树突树的树突状重叠的树木的浦肯野细胞。这样的树突树可以被看作单独邻近的浦肯野细胞树突树重叠时，不表达GFP（ 图1A，B）。 </li></ol> 4。浦肯野细胞树突状树的大小和分支点的测量。 <ol><li>树突状树的大小。与荧光标记的浦肯野细胞的树突状树的一个给定的小区的大小可以很容易地进行测量，如果树与其他细胞中不重叠。由于钙结合蛋白免疫染色标签所有的浦肯野细胞，也可以是难以识别的细胞与非重叠的树突树。在这种情况下，建议使用的PCP2-EGFP，以简化足够的浦肯野细胞的识别s的非重叠的树突树。树突状树与一个非重叠的浦肯野细胞一旦被识别时，它被认为与20倍的镜头，用数码相机拍摄的图像被记录。然后此图像可以与图像分析程序进行分析。我们使用Image Pro Plus中，概述了树突状细胞的树与一个单一的点击鼠标，使用魔术棒工具，在测量模式（ 图2A）。然后程序计算的树突树和覆盖的区域，将其导出到MS Excel中。 GraphPad棱镜软件（见下文）的数据进行统计分析。 </li><li>分支点的数量。由于高度支化和精细形态的浦肯野细胞树突状树的分支点需要手动计算。浦肯野细胞被放大20倍的图像文件，它涵盖了大部分的屏幕使用Adobe Photoshop。每个分支点进行计数，并标有一个亮点（ 图2B）。 </li></ol> 养殖期间监测浦肯野细胞树突状树的发展 来自PCP2-EGFP表达绿色荧光蛋白的小鼠特别是在浦肯野细胞的器官切片文化研究单个细胞的形态在数天中文化。这样的成长和发展养殖期间的树突状树可以很好地记录在案。养殖期间10倍的目标，生活确定的浦肯野细胞进行拍照，每2 或第3 天 。这种低功率的目标选择，以避免和减少光毒性损伤的细胞，由于荧光灯的照明。 图3显示了两个例子，浦肯野细胞拍到在生活文化。所述第一小区其次在体外从2天直到7天在体外 （ 图3，AC）。显而易见的是，dendritiÇ树的这种细胞在这段时间内增加一些新的分支和扩展的大小。在第二个例子中，随后浦肯野细胞的4至11天， 在体外 （ 图3，DF）。在DIV4小的树突状树在这段时间内大幅扩大。这两个例子表明，大多数浦肯野细胞的树突状树出席文化时期的结束的确在文化发展。 发展的浦肯野细胞树突状树是抑制PKC或mGluR1的刺激 器官小脑切片文化培养11天。在体外在第2 次的天开始，任PMA（50纳米），佛波醇酯刺激PKC或DHPG（10μM），mGluR1的激动剂，用一些培养，直到培养物固定。先前已经示出了这两种化合物以抑制和浦肯野细胞的树突状生长7，8，9，5的限制。在未处理的对照切片浦肯野细胞开发了一个典型的大和高度支化的树突状树的可视化可以通过抗钙结合蛋白的免疫染色（ 图4A），或与EGFP表达的浦肯野细胞的培养中，通过与抗-GFP（ 图4D）的免疫染色。文化与PMA处理的树突树的形态，深刻地改变了。树突出现增厚，只有短短的偏科。浦肯野细胞很多，不像在控制文化，不再是单极的，一个主要的枝晶从胞体发出的，但开发两个甚至更多的初级树突（ 图4B，E）。境内的树突状树显着降低（见下文）。类似的情况是目前在DHPG治疗的文化。浦肯野细胞的树突状树被大大缩小尺寸时，和支化显着降低（ 图4C，F）。然而，也有一些qualitat，香港专业教育学院相比差异PMA处理的培养。有没有增厚的树突分支，主要树突进行许多非常短的信号事件在PMA和DHPG治疗的浦肯野细胞可能相似，但不完全相同的二次枝晶（ 图4C，F）。 浦肯野细胞树突状树的大小和分支点进行定量评价的定量评价 ，浦肯野细胞树突树和每浦肯野细胞的分支点的大小是衡量如上所述。从至少三个独立的实验结果汇集起来，并需要最少20细胞以进行分析。对照实验由树突状树木覆盖的面积的平均值来确定和设置为100％，和其他实验的值相应地表示为百分比值。数据的统计分析，使用GraphPad棱镜软件完成。因为我们没有假设所有值的确定是一个高斯分布的一部分，我们采用统计检验不承担这样的分布的测量值。对于比较多的条件和试验统计学意义，我们使用Kruskal-Wallis检验，然后通过适当的程序事后测试等级为基础的统计，因为，例如，实施后唐恩的测试中GraphPad棱镜。结果通常作为条形图。这样的统计评价的一个例子在图5中给出。分析的树突状树的大小（ 图5A）和分支点每蒲肯野细胞的数量（ 图5B），表现出明显的差异之间的控制条件和DHPG或PMA处理。这两个参数有统计学意义，P &lt;0.001根据不同的Kruskal-Wallis检验。 <img alt="图1" src="/files/ftp_upload/3637/3637fig1.jpg" /> FiguRE 1。 EGFP标记的浦肯野细胞。在PCP2-EGFP的小鼠，并不是所有的浦肯野细胞表达EGFP。因此，可以单独观察一些浦肯野细胞的树突状树木时，他们明示EGFP（EGFP通道A中）和相邻的浦肯野细胞树突树重叠不（负EGFP的通道中的A所示，在所示的抗钙结合蛋白染色阳性在B中的红色通道）。比例尺= 50微米。 <img alt="图2" src="/files/ftp_upload/3637/3637fig2.jpg" /> 图2。浦肯野细胞的树突状参数的测量。（A）。的树突状树的大小与一个单一的点击鼠标在图像分析软件Image Pro的加描绘轮廓的浦肯野细胞，用魔术棒工具可以很容易地测量。使用魔术棒工具树突状树，整个包括所有细胞的胞体和树突分支的完全包围而行。 （B）。分支点的数量是凑与指向手动浦肯野细胞的图像。每个分支点都标有一个黄色的点。比例尺= 50微米。 <img alt="图3" src="/files/ftp_upload/3637/3637fig3.jpg" /> 图3。监控发展的浦肯野细胞树突状树。确定的浦肯野细胞，多次被拍到监控的树突树的生长。 （AC）:浦肯野细胞的树突树的成长和发展从第一天开始， 在体外 （DIV）2至DIV7。有持续增长，在这种文化时期的树突的分支。 （DF）:从DIV4浦肯野细胞的树突状树的成长和发展，直到DIV11。树突状树这种文化期间大幅扩大。比例尺= 50微米。 <img alt="图4" src="/files/ftp_upload/3637/3637fig4.jpg" /> 图4。发展的浦肯野细胞树突状树是抑制PKC或mGluR1的stimulati（A），（D）:未经处理的对照组细胞与发达的树突状树。 （B），（E）:经过9天PMA处理树突出现增厚和树突状树强烈减少尺寸。细胞往往失去他们的极化和，成为双极（E）。 （C），（F）:DHPG治疗9天​​后树突树大大减小尺寸。在PMA处理不同，很多很细而短的侧枝上小学和中学的树突存在。细胞往往失去极化，成为双极性（F）。浦肯野细胞抗钙结合蛋白的免疫组化观察（A - C）和EGFP表达的文化来自中PCP2-EGFP的小鼠（D - F）。比例尺= 50微米。 <img alt="图5" src="/files/ftp_upload/3637/3637fig5.jpg" /> 图5。定量评价的浦肯野细胞树突树的大小和分支。定量测量表明，树突树的大小酒吧（上图）和numbePMA或DHPG治疗后r的分支点酒吧（下图）都大大降低。控制树突树和浦肯野细胞的树突树处理文化之间的差异显着性，P &lt;0.001。

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动物进行了实验，根据欧洲共同体理事会1986年11月24日的指令"（86/609/EEC）进行了审查和瑞士当局允许的。作者有没有透露。

这里介绍的方法允许研究器官小脑切片培养和浦肯野细胞的树突状发展，定量地评价测定树突状树的尺寸和数目的树突状分支点的浦肯野细胞的树突状扩展。当然，一个更广泛，更复杂的定量分析，浦肯野细胞树突是可能的，例如，通过测定总树突长度，执行一个Sholl的分析，确定分形维数的树突状树。对于这种类型的分析，它通常需要手动跟踪整个树的分析程序，例如神经龙力浦肯野细胞树突状。虽然这些更精细的方法当然产生了高水准的分析（例如，见1，6），他们花费更多的时间，需要更专业的设备和分析软件相比，这里介绍的方法。对于大多数应用，在特别的情况下，变更在相当大的质和可见的浦肯野细胞树突，提出了简单的方法就足够了。应该注意的是，来自P8小鼠小脑的器官切片文化将只包括浦肯野细胞树突发育的后期阶段特征的快速树突状扩张。为了进行这项研究的早期阶段，片培养的方法需要能够适应从新生儿或胚胎的动物的切片，然后将树突发育的早期阶段（如2）。 器官小脑浦肯野细胞切片文化的可视化，我们只集中在两个常规方法:反钙结合蛋白的免疫组化和浦肯野细胞的小鼠表达。当然，更多的方法，特别是基因枪或病毒转染荧光蛋白，diolistic用荧光染料标记和细胞内染料注射到单一的浦肯野细胞。根据要求的研究，并在实验室的设备中可用的这些方法可以显着提高的浦肯野细胞的树突状形态的水平的分析，例如，通过使用共聚焦或两个光子显微镜分析树突棘。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>组织培养插入</td><td>密理博</td><td> PICM 03050 PICM ORG50 </td><td> PICMORG50插入低的边缘，并允许在显微镜观察活的文化</td></tr><tr><td> MEM </td><td> Gibco公司，Invitrogen公司</td><td> 11012044 </td><td></td></tr><tr><td> Glutamax 1 </td><td> Gibco公司，Invitrogen公司</td><td> 35050038 </td><td></td></tr><tr><td>基底Medum鹰</td><td> Gibco公司，Invitrogen公司</td><td> 41010026 </td><td></td></tr><tr><td>马血清</td><td> Gibco公司，Invitrogen公司</td><td> 26050070 </td><td></td></tr><tr><td>佛12-myrista的特13 - 乙酸酯（PMA） </td><td> TOCRIS </td><td> 1201 </td><td></td></tr><tr><td> （RS）-3,5 - 二羟基-苯基甘氨酸（DHPG） </td><td> TOCRIS </td><td> 0342 </td><td></td></tr><tr><td>兔抗钙结合蛋白D-28K </td><td> Swant </td><td> CB38 </td><td></td></tr><tr><td>反NEUN，克隆A60 </td><td> Chemicon公司，密理博</td><td> MAB377 </td><td></td></tr><tr><td>兔抗GFP </td><td> Abcam公司</td><td> Ab290 </td><td></td></tr></tbody></table>

the analysis of purkinje cell dendritic morphology in organotypic slice cultures

浦肯野细胞是一个有吸引力的模型系统，研究树突状发展，因为他们有一个令人印象深刻的树突状树是严格面向在矢状面和发展主要是在产后期在小型啮齿动物3。此外，几种抗体可用选择性和集中标签包括所有进程的浦肯野细胞，用抗钙结合蛋白Calbindin D28K被最广泛使用的。活细胞的树突观看，都可以通过杰克逊实验室小鼠表达绿色荧光蛋白选择性地在浦肯野细胞11。器官小脑片培养细胞可以轻松的浦肯野细胞树突发育的实验操作，因为大多数的浦肯野细胞树突树的树突状扩张过程中实际发生的培养周期4。我们在座的短，可靠和简单的协议，用于观察和分析浦肯野细胞的树突状形态，生长在organotypiÇ小脑切片文化。出于多种目的，定量评价的浦肯野细胞树突状树是可取的。我们关注两个参数，树突树的大小和分支点的电话号码，可以快速，轻松地确定反小脑钙结合蛋白染色切片文化。这两个参数产生变化的浦肯野细胞树突状树一个可靠的和敏感的措施。使用的例子，治疗与蛋白激酶C（PKC）激活剂PMA和代​​谢型谷氨酸受体1（mGluR1的），我们演示了如何在树突发育的差异可视化和定量评估。的存在下的结合，一个广泛的树突状树，选择性和强烈的免疫染色的方法，覆盖的枝晶生长期间和小鼠模型的浦肯野细胞的特定的EGFP表达，使树突状揭示的机制的浦肯野细胞的强大的模型系统的器官型切片培养发展。

Watch this Scientific Journal Video about The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures at JoVE.com

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

我们提出了一个协议，允许查看和定量驴个别浦肯野细胞的树突树的生长在器官小脑切片文化形态。该协议的目的是促进研究的浦肯野细胞树突发育的机制。

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

the-analysis-purkinje-cell-dendritic-morphology-organotypic-slice

Nanyang Technological University

Research

JoVE Journal

Neuroscience

18.5K 次观看.  University of Basel. 我们提出了一个协议，允许查看和定量驴个别浦肯野细胞的树突树的生长在器官小脑切片文化形态。该协议的目的是促进研究的浦肯野细胞树突发育的机制。

视频: The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

神经科学

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

脊髓 Cocultures 丘脑-皮质轴突分支和突触形成的可视化研究

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

髓鞘有机小脑片培养的制备及免疫染色

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

器官细胞生存在器官细胞切片培养

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

<meta charset="utf8"></head><body>小脑浦肯野细胞:器官型培养物中的 DNA 损伤反应

Visualizing the DNA Damage Response in Purkinje Cells Using Cerebellar Organotypic Cultures

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional activity, making them particularly vulnerable to DNA damage. This necessitates an efficient DNA damage response (DDR) to prevent cerebellar degeneration, often initiated by PC dysfunction or loss. A notable example is the genome instability syndrome, ataxia-telangiectasia (A-T), marked by progressive PC depletion and cerebellar deterioration. Investigating DDR mechanisms in PCs is vital for elucidating the pathways leading to their degeneration in such disorders. However, the complexity of isolating and cultivating PCs in vitro has long hindered research efforts. Murine cerebellar organotypic (slice) cultures offer a feasible alternative, closely mimicking the in vivo tissue environment. Yet, this model is constrained to DDR indicators amenable to microscopic imaging. We have refined the organotypic culture protocol, demonstrating that fluorescent imaging of protein-bound poly(ADP-ribose) (PAR) chains, a rapid and early DDR indicator, effectively reveals DDR dynamics in PCs within these cultures, in response to genotoxic stress.

<meta charset="utf8"></head><body>作者聚焦:破译 ATM 在共济失调-毛细血管扩张症和相关小脑变性中的作用

Cerebellar Purkinje cells (PCs) are particularly sensitive to deficiencies in the DNA damage response. A protocol is presented for the visual evaluation of the dynamics of PC response to DNA damage, which involves staining protein-bound poly(ADP-ribose) chains within cerebellar organotypic cultures.