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。

<ol>
	<li>Bosman, L. W., Hartmann, J., Barski, J. J., Lepier, A., Noll-Hussong, M., Reichardt, L. F., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+TrkB+for+synapse+elimination+in+developing+cerebellar+Purkinje+cells.">Requirement of TrkB for synapse elimination in developing cerebellar Purkinje cells.</a> Brain Cell Biol. 35, 87-101 (2006).</li><li>Boukhtouche, F., Janmaat, S., Vodjdani, G., Gautheron, V., Mallet, J., Dusart, I., Mariani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoid-related+orphan+receptor+alpha+controls+the+early+steps+of+Purkinje+cell+dendritic+differentiation.">Retinoid-related orphan receptor alpha controls the early steps of Purkinje cell dendritic differentiation.</a> J. Neurosci. 26, 1531-1538 (2006).</li><li>Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+control+of+dendritic+growth+and+development+of+cerebellar+Purkinje+cells.">Cellular and molecular control of dendritic growth and development of cerebellar Purkinje cells.</a> Prog. Histochem. Cytochem. 39, 131-182 (2004).</li><li>Kapfhammer, L. C., Doering, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+slice+cultures.">Cerebellar slice cultures.</a> Protocols for Neural cell culture. , 285-298 (2010).</li><li>Gugger, O. S., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+size+of+the+dendritic+tree+does+not+protect+Purkinje+cells+from+excitotoxic+death.">Reduced size of the dendritic tree does not protect Purkinje cells from excitotoxic death.</a> J. Neurosci. Res. 88, 774-783 (2010).</li><li>Li, J., Gu, X., Ma, Y., Calicchio, M. L., Kong, D., Teng, Y. D., Yu, L., Crain, A. M., Vartanian, T. K., Pasqualini, R., Arap, W., Libermann, T. A., Snyder, E. Y., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mediates+Purkinje+cell+dendritic+development+via+lysyl+oxidase+propeptide+and+NF-&#954;B+signaling.">mediates Purkinje cell dendritic development via lysyl oxidase propeptide and NF-&#954;B signaling.</a> Neuron. 68, 45-60 (2010).</li><li>Metzger, F., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+activity+modulates+dendritic+differentiation+of+rat+Purkinje+cells+in+cerebellar+slice+cultures.">Protein kinase C activity modulates dendritic differentiation of rat Purkinje cells in cerebellar slice cultures.</a> Eur. J. Neurosci. 12, 1993-2005 (2000).</li><li>Schrenk, K., Kapfhammer, J. P., Metzger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+dendritic+development+of+cerebellar+Purkinje+cells+in+slice+cultures+from+protein+kinase+C?-deficient+mice.">Altered dendritic development of cerebellar Purkinje cells in slice cultures from protein kinase C?-deficient mice.</a> Neuroscience. 110, 675-689 (2002).</li><li>Sirzen-Zelenskaya, A., Zeyse, J., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+class+I+metabotropic+glutamate+receptors+limits+dendritic+growth+of+Purkinje+cells+in+organotypic+slice+cultures.">Activation of class I metabotropic glutamate receptors limits dendritic growth of Purkinje cells in organotypic slice cultures.</a> Eur. J. Neurosci. 24, 2978-2986 (2006).</li><li>Stoppini, L., Buchs, P. -. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J. Neurosci. Methods. 37, 173-182 (1991).</li><li>Tomomura, M., Rice, D. S., Morgan, J. I., Yuzaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Purkinje+cells+by+fluorescence-activated+cell+sorting+from+transgenic+mice+that+express+green+fluorescent+protein.">Purification of Purkinje cells by fluorescence-activated cell sorting from transgenic mice that express green fluorescent protein.</a> Eur. J. Neurosci. 14, 57-63 (2001).</li><li>Weyer, A., Schilling, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+cell+type-specific+expression+of+the+neuronal+marker+NeuN+in+the+murine+cerebellum.">Developmental and cell type-specific expression of the neuronal marker NeuN in the murine cerebellum.</a> J. Neurosci. Res. 73, 400-409 (2003).</li></ol>

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?...

<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

Research

JoVE Journal

Neuroscience

18.4K 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

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

科研

神经科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

器官型小脑文化：细胞凋亡的挑战和检测

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

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 ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

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 ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.