我们感谢N. Schwedhelm - 杜美尔优秀的技术援助，三锤和S. Papiol帮助与统计分析。我们的工作是由马普学会，德意志研究联合会，该中心的纳米显微镜，及脑分子生理学（CNMPB），哥廷根，德国和由GGNB初级组助学金哥廷根大学的资助。

小脑颗粒神经元可以从产后当天（P）5小鼠幼崽或P6幼鼠准备。我们遵循的协议，由Bilimoria医师和他的同事描述的，它采用了有丝分裂抑制剂，以选择用于有丝分裂后小脑颗粒神经元13。 伦理语句： 根据批准的“Verbraucherschutz UND Lebensmittelsicherheit”下萨克森州，德国的动物协议所有涉及活的动物实验已经进行的。 体外测定： 1。对于磷酸钙转染方法制备的DNA质粒，媒体，和缓冲器的<ol><li>溶解质粒DNA在无菌，无热原的水; DMEM（高糖），使250万氯化钙 ;使2X HBSS（将4克氯化钠，0.1775克 ​​氯化钾，0.095克的Na 2 HPO 4•7H 2 O，0.675克葡萄糖和250毫升的超纯水2.5克HEPES和调节pH值至7.05，7.08，和7.11）。注：当准备2倍HBSS溶液，测试其pH值提供了有关质粒的特定组合的转染效率达到最佳效果。 </li></ol> 2。培养神经细胞的转染<img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig1highres.jpg" src="/files/ftp_upload/51070/51070fig1.jpg" /> 图1。 体外轴突和树突生长测定法的流程图。培养小脑颗粒神经元（24孔板用玻璃盖玻片），分离出P6幼鼠，转染在DIV 0或1，含有荧光标记的转染（ 如 GFP）的DNA沉淀。固定和免疫后，神经元的成像采用盲法的方式。影像会导入的ImageJ和流程计量。测量，然后用统计软件进行处理。 <ol><li>种子小脑颗粒神经元（20×10 6每24孔板; BME，10％小牛血清，2mM青霉素-Streptomycin-谷氨酰胺（PSG），25mM的KCl）中的硝酸洗涤，聚鸟氨酸涂布的12毫米盖玻片的24孔板用500μl的媒体，每孔。 </li><li>一天在体外 （DIV）0（至少8小时后镀）或DIV 1，收集培养基，并保持在37°C用500μl预热的DMEM洗两次神经元和加入500μlDMEM中。 </li><li>在孵化广场神经元（37℃，5％CO 2）45分钟。 </li><li>准备40微升DNA沉淀每个孔的混合：脱氧核糖核酸（2-2.5微克/孔，总DNA的10％应该是一个转染标记如 GFP可视化转染神经元），水（最多18微升），加2微升2.5M的氯化钙 ，拌匀，加入20微升2倍的HBSS。 <ol><li>孵育DNA沉淀5分钟，在RT。 </li></ol></li><li>添加DNA沉淀到各孔中，孵育神经元在孵化18分钟。 </li><li>删除的DMEM / DNA混合物，并用500μl预热的DM洗两次神经元EM。 </li><li>从步骤2.2添加回收集媒体的神经元。如果神经元将在培养3天以上，补充媒体25毫米的葡萄糖在DIV 3补充碳源。 </li><li>经过1-5天，受神经元免疫细胞化学利用绿色荧光蛋白的抗体。 </li><li>图像的至少30％的条件下的单个神经元中使用荧光显微镜以盲方式。 </li></ol> 3。测量轴突和树突与NeuronJ，一个国家卫生研究院ImageJ的插件重要提示：请确保图像通过使用适当的像素correctluy规模：微米比例视放大率和图像的分辨率。 <ol><li>图像转换为8位与ImageJ的：打开图像，选择“图像” - &gt;“类型” - &gt; &#39;8位&#39; - &gt;&#39;保存&#39;的形象。 </li><li>运行NeuronJ插件并打开8位图像。 <img alt="屏幕截图" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot1.jpg" width="500px" /></li><li>使用“添加描记&#39;选项来跟踪轴突：CLICK鼠标左键一次是在轴突的开始，移动鼠标沿的过程。双击轴突的尖端，如果跟踪轴突形状相匹配。 <img alt="屏幕截图" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot2.jpg" width="500px" /> 注意：如果建议跟踪从轴突形状不同，单击一次轴突过程锚跟踪，然后双击轴突的尖端。 </li><li>点击“测量描记”，选择“显示跟踪测量”选项，然后按“运行”。轴突的测量都显示在一个新窗口。对于树突，选择“显示组的测量选项”，然后按“运行”。 <img alt="屏幕截图" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot3.jpg" width="500px" /> 总枝晶的测量都显示在一个新窗口。将其保存为可以在任何电子表格程序中打开一个单独的文件。 </li><li>或者手动跟踪，使用斐济软件：右键单击日E&#39;直线&#39;选项，选择“手绘线”， <img alt="屏幕截图" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot4.jpg" width="500px" /> 保持按下鼠标左键并手动跟踪过程中，按“CTRL + M”来衡量。 </li><li>计算每个条件平均轴突/树突长度和使用适当的统计检验。 体内电穿孔： </li></ol> 1。设备和试剂的制备<ol><li>你需要30 G针，间隔（1-2毫米），注射器，死体积减速机（DVR），电穿孔和tweezertrodes，加热垫或红外线保温灯，鹅颈灯和异氟醚。 </li><li>把DVR成针，然后将针注射器，最后把垫片针上（ 图2）。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig2highres.jpg" src="/files/ftp_upload/51070/51070fig2.jpg" /> 数字2。针。DVR的制剂被切断200μl的移液管尖端，并置于针以减少死体积。垫片是从200微升加载提示衍生和被放置在针的末端，以调节渗透深度进入小脑到大约2毫米。标尺单位：厘米</li><li>在PBS/0.03％固绿溶解DNA。注意：作为一个转染的标记，但有利的是使用一种荧光蛋白质，是下一个神经元特异性启动子（ 例如，突触蛋白），以唯一的可视化的神经元。总质粒量的25％应是编码转染标记的质粒上。 </li><li>使70％的乙醇。 </li><li>混合十月等体积和30％的蔗糖溶解在PBS中。 </li><li>填充注射器4微升的DNA（4微克/微升的PBS/0.03％固绿质粒DNA）的。 </li></ol> 2。幼鼠的香港专业教育学院 香港专业教育学院的流程图：参见图3 图3。 体内电穿孔。P4幼鼠流程图被麻醉的异氟醚和质粒DNA编码荧光标记物的转染（ 如 GFP）被注入到小脑，随后暴露于5电脉冲。五天后，分离GFP阳性小脑被切片，进行免疫组织化学染色。图像是使用共焦显微镜拍摄，并使用了Imaris软件进行分析。数据处理与统计程序。 <ol><li>从白化菌株（只Wistar或长埃文斯）使用P4的幼鼠。 </li><li>麻醉幼崽（一个接一个）与异氟醚的小盒子（ 例如 P1000的枪头盒）与200微升异氟醚（浸泡在组织）1-2分钟，直到小狗不再移动。请小心，幼崽不要接触瓦特第i个Iiquid异氟醚。监视时间密切个别幼犬麻醉的反应不同。 </li><li>消毒背部小狗的头，用70％的乙醇。 </li><li>修复拇指和食指之间的小狗的头，用鹅颈灯来定位白化小狗的小脑。横窦大幅标定中脑（上，下丘）从皮质半球（ 图3）。小脑毗邻脑并出现在暗的阴影。使用永久性标记，以指示以点小脑。重要提示：请小狗在固定的位置！注：如果麻醉在此过程中磨掉，露出小狗到异氟烷注射的DNA之前。 </li><li>插入针（ 图3），慢慢注入3μl的DNA的插入小脑。 </li><li>让DNA溶液扩散为30-60秒。 <ol><li>将tweezertrodes之间小狗的头，使该负极使用后脑勺（小脑接触R区域），而正极接触头的相对侧（ 图3）。 </li><li>待小狗到5的电脉冲。调整电压，以重幼崽，以确保良好的电效率，而不影响其生存期（ 表1）。 <table border="0"><tbody><tr><td>重量</td><td>电压</td><td>脉冲</td><td>间隔</td></tr><tr><td> 8-9克</td><td> 160 V </td><td> 50毫秒</td><td> 950毫秒</td></tr><tr><td> 9-10克</td><td> 165 V </td><td> 50毫秒</td><td> 950毫秒</td></tr><tr><td> &gt;10克</td><td> 170V的</td><td> 50毫秒</td><td> 950毫秒</td></tr></tbody></table> 表1中。电穿孔的P4幼鼠。 </li><li>让幼犬恢复上加热垫或红外线灯的下方。返回幼仔大坝。重要提示：请确保加热源不造成任何灼伤。 </li></ol></li><li>通过将它们的CO 2，然后斩首电5天后牺牲幼崽。 <ol><li>隔离小脑和屏幕的GFP阳性的人小脑用荧光显微镜。 </li></ol></li><li>小脑固定在4％PFA O / N在4℃，然后在4℃孵育在30％蔗糖，直到小脑下沉到试管底部。 </li><li>嵌入小脑在OCT/30％蔗糖和切为40μm冠状切片用低温恒温器。注：在一项双盲的方式各节小脑。 </li><li>主题部分，以免疫组化用绿色荧光蛋白抗体。染液核染料（DAPI或赫斯特33258），并确定每只动物至少200转染的神经元的观察。 </li><li>供了深入的分析，细分IGL成两半，从而导致面向ML和低级IGL对着白质的上部IGL和计数驻留在每个半GFP阳性神经元。注：计数以盲方式各部分的GFP阳性神经元。 </l我&gt; </ol> 3。测量枝蔓长，采集的部分在X，Y，Z平面的图像使用共聚焦显微镜注：例如，使用40张为40μm的部分与Z-stwp为1微米。 <ol><li>打开的图像序列中的软件，Imaris里，产生枝晶的3D图像。 </li><li>点击&#39;超越&#39;的模式来检视神经元在三维。 <img alt="屏幕截图" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot5.jpg" width="500px" /></li><li>选择“添加新的灯丝”，然后点击“跳过自动创建&#39;开始半自动跟踪。 <img alt="屏幕截图" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot6.jpg" width="500px" /> 注：分析在盲法每个3D图像</li><li>选择“绘图”选项卡和“AutoPath”。 <img alt="屏幕截图" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot7.jpg" width="500px" /></li><li>移动对C的鼠标光标埃尔身体和Shift +鼠标右键点击，选择细胞体。注：Autocalculation软件可能需要几分钟。 <img alt="屏幕截图" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot8.jpg" width="500px" /></li><li>使用Shift键+鼠标左键点击路径添加到灯丝（树突）。注意：路径可以实时可视化。 <img alt="屏幕截图" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot9.jpg" width="500px" /></li><li>去长丝统计窗口，点击“详细”，“特殊值”和“丝枝晶长度（和）&#39;的枝蔓总长度的总和。 <img alt="屏幕截图" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot10.jpg" width="500px" /></li><li>使用适当的统计测试，以分析数据。 </li></ol>

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J., Schwedhelm-Domeyer, N., Nakazawa, T., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p250GAP+is+a+novel+player+in+the+Cdh1-APC/Smurf1+pathway+of+axon+growth+regulation.">p250GAP is a novel player in the Cdh1-APC/Smurf1 pathway of axon growth regulation.</a> PLoS One. 7, (2012).</li><li>Kannan, M., Lee, S. J., Schwedhelm-Domeyer, N., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+E3+ligase+Cdh1-anaphase+promoting+complex+operates+upstream+of+the+E3+ligase+Smurf1+in+the+control+of+axon+growth.">The E3 ligase Cdh1-anaphase promoting complex operates upstream of the E3 ligase Smurf1 in the control of axon growth.</a> Development. 139, 3600-3612 (2012).</li><li>Gaudilliere, B., Konishi, Y., de la Iglesia, N., Yao, G., Bonni, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII-NeuroD+Signaling+Pathway+Specifies+Dendritic+Morphogenesis.">CaMKII-NeuroD Signaling Pathway Specifies Dendritic Morphogenesis.</a> Neuron. 41, 229-241 (2004).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cdc20-APC+ubiquitin+signaling+pathway+regulates+presynaptic+differentiation.">A Cdc20-APC ubiquitin signaling pathway regulates presynaptic differentiation.</a> Science. 326, 575-578 (2009).</li><li>Litterman, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+OBSL1-Cul7Fbxw8+ubiquitin+ligase+signaling+mechanism+regulates+Golgi+morphology+and+dendrite+patterning.">An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning.</a> PLoS Biol. 9, (2011).</li><li>Kim, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+centrosomal+Cdc20-APC+pathway+controls+dendrite+morphogenesis+in+postmitotic+neurons.">A centrosomal Cdc20-APC pathway controls dendrite morphogenesis in postmitotic neurons.</a> Cell. 136, 322-336 (2009).</li><li>Shalizi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calcium-regulated+MEF2+sumoylation+switch+controls+postsynaptic+differentiation.">A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation.</a> Science. 311, 1012-1017 (2006).</li><li>Vadhvani, M., Schwedhelm-Domeyer, N., Mukherjee, C., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Centrosomal+E3+Ubiquitin+Ligase+FBXO31-SCF+Regulates+Neuronal+Morphogenesis+and+Migration.">The Centrosomal E3 Ubiquitin Ligase FBXO31-SCF Regulates Neuronal Morphogenesis and Migration.</a> PLoS One. 8, (2013).</li><li>Puram, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CaMKIIbeta+signaling+pathway+at+the+centrosome+regulates+dendrite+patterning+in+the+brain.">A CaMKIIbeta signaling pathway at the centrosome regulates dendrite patterning in the brain.</a> Nat. Neurosci. , (2011).</li><li>Jia, Y., Zhou, J., Tai, Y., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC+channels+promote+cerebellar+granule+neuron+survival.">TRPC channels promote cerebellar granule neuron survival.</a> Nat. Neurosci. 10, 559-567 (2007).</li><li>Huynh, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isoform-specific+SnoN1-FOXO1+repressor+complex+controls+neuronal+morphogenesis+and+positioning+in+the+mammalian+brain.">An isoform-specific SnoN1-FOXO1 repressor complex controls neuronal morphogenesis and positioning in the mammalian brain.</a> Neuron. 69, 930-944 (2011).</li><li>de la Torre-Ubieta, L., Bonni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+neuronal+polarity+and+morphogenesis+in+the+mammalian+brain.">Transcriptional regulation of neuronal polarity and morphogenesis in the mammalian brain.</a> Neuron. 72, 22-40 (2011).</li><li>Calissano, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+insulin-like+growth+factor+I+exerts+a+trophic+action+and+confers+glutamate+sensitivity+on+glutamate-resistant+cerebellar+granule+cells.">Recombinant human insulin-like growth factor I exerts a trophic action and confers glutamate sensitivity on glutamate-resistant cerebellar granule cells.</a> Proc. 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作者宣称没有竞争的财务权益。

优点和在体外和体内方法所描述的限制： 从小鼠和大鼠培养的小脑颗粒神经元也同样适用以及形态学分析。由于一只老鼠小脑的大尺寸，CNGS从幼鼠的产量超过了小鼠幼崽3至4倍。除小脑颗粒神经元，皮质和海马神经元可被用作培养系统为好。磷酸钙方法导致低（0.01-5％）的转染效率，这是需要的，分析单个神经元的形态。替代亲脂转染方法可以用于为好，但过?...

小脑是一个很好的系统来研究轴突生长和迁移的机制。自1神经科学的曙光小脑已经解剖研究的主题。现代显微镜和免疫组化技术已显著扩大，由圣地亚哥，拉蒙和卡哈尔2-4细化初始的发现。小鼠遗传学和分子生物学研究发现重要的增长和转录因子在小脑发育的控制，从而导致需要对不同类型的神经元，包括小脑颗粒神经元（小脑颗粒神经元）5-7的正确接线关键事件的更深入的了解。 小脑是显影后脑8的菱1的衍生物。菱形唇，它是4 个脑室的屋顶的一部分，引起的小脑颗粒神经元祖细胞，这将构成最大量的神经元群中的成人小脑9。下面的喙迁移，他们定居在小脑原基。在这里，颗粒神经元前体有丝分裂导致外颗粒层（EGL），其中发生在出生后啮齿动物的急剧扩张。来自东瀛，神经元开始通过分子层（ML）向内迁移，过去的浦肯野细胞层，最终定居在内部颗粒层（IGL 2）。在这个迁徙的过程中，他们获得一个双极形状有两个轴突延伸到ML。在进一步迁移，细胞体迁移远离轴突和两个进程融合以形成一个分叉的，T形的轴突10。随后，这些轴突束状和被称为平行纤维。已入驻IGL，小脑颗粒神经元树突生长，形成树枝状的爪子，建立突触苔藓纤维。审查基本过程中的显影小脑，组合在体外和体内 approach允许进行可靠的结果和结论。 小脑颗粒神经元不仅是小脑而是整个大脑的最大量的神经元，并且可以被培养，以高纯度11-13。在文化方面，这个高度同质化的神经元群变得迅速和有丝分裂后获得一个极形态，易于识别轴突和树突。培养的小脑颗粒神经元已被证明是研究神经发育的各个方面，包括祖细胞增殖，分化，轴突和树突发育，神经细胞迁移，凋亡及电生理特性（14-19和许多其他人）非常有用。利用基因操作的扩大培养的小脑颗粒神经元的通用性，并允许进行进一步机械洞察上述事件。使用低效率的磷酸钙或亲脂性的方法，其次是免疫细胞化学与极性标记或软件支持的分析facili培养神经元的转染塔特斯单个神经元，例如在一个密集的神经元文化形态的评估。用这种方法，在轴突或枝晶生长兴趣蛋白质的作用，可以研究20-25,26-28。然而，这种文化体系是分析神经细胞迁移的迁移是非常有限的高密度培养的那么有用，并需要共同培养。 在体外分析轴突和树突生长还允许一个信号传导途径的利用RNA干扰（I）的组合相互连接的蛋白质的检测，过度表达或小分子。 建立在轴突和树突生长调控或神经细胞迁移的目的蛋白的相关性， 在体内电穿孔（IVE）技术允许用于分析在显影小脑皮层。由于其在啮齿类动物小脑发育延伸的方式进入第一产后2周的事实，小脑表示accessib乐大脑结构的遗传操作研究发展轴突和树突，神经元迁移，突触和凋亡20-24,29,30,26,27,31-34。此外，该模型体系也为神经元发育的需要完整的小脑皮层如轴突路径，布线和神经元和神经胶质细胞的相互作用两者合计的连接等方面有用的，该协议提供了在体外和体内技术来解决有关神经元的形态发生和迁移互补的方法。

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			<td style="width:125px;">Gibco</td>
			<td style="width:88px;">11960-044</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">BME</td>
			<td style="width:125px;">Gibco</td>
			<td style="width:88px;">41010-026</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Insulin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:88px;">Si-1-4011</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Poly-L-Ornithine</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:88px;">P-2533</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:160px;">CaCl2</td>
			<td style="width:125px;">Appli-Chem</td>
			<td style="width:88px;">A3652</td>
			<td style="width:168px;"></td>
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			<td height="42" style="height:42px;width:160px;">Isofluorane</td>
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			<td height="22" style="height:22px;width:160px;">Tissue-Tek OCT</td>
			<td style="width:125px;">Sakura</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:160px;">Material Name</td>
			<td style="width:125px;">Company</td>
			<td style="width:88px;">Catalogue Number</td>
			<td style="width:168px;">Comments (optional)</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:160px;">ECM 830 and tweezertrodes</td>
			<td style="width:125px;">Harvard Apparatus</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:160px;">Epifluorescence microscope and camera</td>
			<td style="width:125px;">Nikon</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:160px;">SP2 confocal microscope</td>
			<td style="width:125px;">Leica</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">ImageJ</td>
			<td style="width:125px;">NIH</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">Imaris 7.4.2</td>
			<td style="width:125px;">Bitplane, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:160px;">GraphPad Prism</td>
			<td style="width:125px;">GraphPad Software, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">MS Excel</td>
			<td style="width:125px;">Microsoft</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
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			<td height="22" style="height:22px;width:160px;">Loading tip 1-200 &#181;l</td>
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			<td style="width:88px;">4853</td>
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			<td height="22" style="height:22px;width:160px;">Pipette tip 200 &#181;l</td>
			<td style="width:125px;">Sarstedt</td>
			<td style="width:88px;">70.760.502</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:160px;">Microlance 3 needle, 30 gauge</td>
			<td style="width:125px;">BD</td>
			<td style="width:88px;">302200</td>
			<td style="width:168px;"></td>
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			<td height="43" style="height:43px;width:160px;">50 &#181;l gastight Syringe 1705</td>
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			<td height="43" style="height:43px;width:160px;">Glass coverslips&#160;</td>
			<td style="width:125px;">Thermo Scientific Menzel Glaeser</td>
			<td>CB00120RA1</td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration

在大脑发育事件包括神经元的形态发生和迁移是高度协调的过程， 在体外和体内分析，以便作深入刻画，以确定参与这些活动的途径。小脑颗粒神经元（小脑颗粒神经元），这些来自发展中国家小脑派生是一个理想的模型系统，允许进行形态学分析。在这里，我们描述了如何将遗传操纵小脑颗粒神经元的方法，以及如何学习axono和树突单个神经元。用这种方法RNA干扰，过表达或小分子的效应可以比较来控制神经元。另外，啮齿动物小脑皮层是体内系统，由于其优越的出生后发育的方便。我们还提出了一个在体内电穿孔技术，基因操作的开发小脑，并说明后续小脑分析评估神经元形态的ND迁移。

分析小脑颗粒神经元的响应不同培养条件的形态，我们转染对DIV 0的神经元，如上所述。转染后，我们把一组神经元的充分培养基（BME，10％小牛血清，2mM PSG，25mM的KCl）中，另一组为最小的含有培养基的胰岛素（BME，25mM的葡萄糖，2mM的PSG，10微克/毫升胰岛素）。我们使用GFP抗体在DIV 1,2进行的神经元免疫细胞化学，和3，随后通过测量轴突和树突为组1和轴突只为一组2。由于血清和KCl，提供生长因?...

Watch this Scientific Journal Video about Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration at JoVE.com

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

神经元的形态发生和迁移的基本正常的大脑发育至关重要的事件。在这里，我们描述的方法来操纵基因培养的小脑颗粒神经元和小脑发展的形态和神经元的迁徙特性的评估。

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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Neuroscience

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

13.6K 次观看.  Max Planck Institute of Experimental Medicine. 神经元的形态发生和迁移的基本正常的大脑发育至关重要的事件。在这里，我们描述的方法来操纵基因培养的小脑颗粒神经元和小脑发展的形态和神经元的迁徙特性的评估。 

视频: Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

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

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

鼠神经母细胞核转染到研究神经细胞迁移<em&gt;在体外</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

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 ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...