这项工作是支持部分由一个生物医学布希批准，美国国家科学基金会资助IBN - 0548543，美国国家科学基金会资助IBN - 0919747，优生优育基金会基金会赠款1 - 2004财年的107的3月，三月优生优育基金会基金会赠款1 - 2008财年的464（BLF）。由美国国立卫生研究院生物技术培训津贴T32 GM008339 - 20 MKK和CGL的支持，CGL的是由新泽西州委员会也支持脊髓研究Predoctoral奖学金08 - 2941 - SCR - E - 0。

1。开始之前： 1）E18大鼠夹层： E18海马神经元的标准清扫方法以前曾描述 13 。为了使用篝火方案的突起的形态特征进行分析，8位。TIF的单个神经元的图像，必须获得。这可以实现的方式取决于你下面的实验协议。神经元可镀在一个足够低的密度，使单个神经元出现在显微镜领域。另外，图像，是生长在一个密集的文化的单个神经元，神经元可转用一种荧光蛋白的质粒编码的各种转染方法。 2）软件要求及安装： <ol class="ualpha"><li>从美国国立卫生研究院和NeuronStudio ImageJ软件NeuronJ插件必须都安装才能运行的篝火方案。软件包可在以下网站找到： ImageJ - <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> NeuronJ - <a href="http://www.imagescience.org/meijering/software/neuronj/">http://www.imagescience.org/meijering/software/neuronj/</a> NeuronStudio - <a href="http://research.mssm.edu/cnic/tools-ns.html">http://research.mssm.edu/cnic/tools-ns.html</a> </li><li>可以发现在http://lifesci.rutgers.edu/〜firestein篝火方案。篝火用户手册，也可在本网站。此外，篝火最初的描述，可以发现在翰墨2010年，流式细胞仪等。我们所有的数据进行了分析，使用的是Windows操作系统。 </li></ol> 3）图像分辨率调节： 您将需要调整的基础上要分析的图像，图像分辨率的篝火方案。在篝火计划bonfire_parameters的一部分，取代当前值与您的影像（微米/像素）的图像分辨率值的变量pix_conv。 2。文件的结构： 为了篝火来分析数据，文件必须是有组织的在这个特定的结构（ 图2）。您将拥有： <ul><li>一个主文件夹</li><li>子文件夹（包含您的每一个不同的条件） </li><li>在不同条件下的文件夹中包含细胞图像文件（filename.tif文件） </li><li>篝火文件夹中包含的篝火MATLAB mfiles（也包含在主文件夹） </li></ul> 3。 NeuronJ跟踪神经元： <ol><li>准备跟踪图像<ol class="lalpha"><li>打开NeuronJ工具栏上选择“打开”按钮，选择你想跟踪的图像图像。 </li><li>选择“最大化”按钮的大小的图像。 </li><li>调整图像的亮度和对比度，让你可以想像NeuronJ工具栏上选择“图像”，然后选择“调整亮度/对比度”的突起。 </li></ol></li><li>在NeuronJ跟踪的细胞体，并确定为“06型”跟踪<ol class="lalpha"><li>选择“添加痕迹”的NeuronJ工具栏上的按钮。 </li><li>跟踪周围的细胞体的周长。 </li><li>选择“标签追查”上的NeuronJ工具栏上的按钮。 </li><li>从“跟踪ID”下拉菜单中选择“N1”。 </li><li>类型选择“06”在NeuronJ：属性窗口，选择“OK”。 </li></ol></li><li>跟踪NeuronJ的突起<ol class="lalpha"><li>选择“添加痕迹”的NeuronJ工具栏上的按钮。 </li><li>添加每个突起的分支沿痕迹。您可以选择只树突或轴突，这取决于您的实验。 </li><li>我们建议，你画的段停留在每个分支点，每个女儿的分支点，在这一点上开始一个新的跟踪。 </li><li>选择NeuronJ工具栏上的“保存描”按钮。 </li><li>保存跟踪你刚才创建在同一文件夹中的原始图像文件。 </li></ol></li><li>从NeuronJ导出的跟踪文件和跟踪标识符文件 两种类型的文件，必须从NeuronJ出口和到相应的条件文件夹一起保存。tif和NDF文件。 <ol class="lalpha"><li> NeuronJ工具栏上选择“导出跟踪”按钮。 </li><li>选择“deliminated标签文本文件：每个跟踪”选项“NeuronJ单独的文件：导出”对话方块，选择“OK”。 </li><li>允许NeuronJ选择的文件的名称和保存位置。 </li><li>选择NeuronJ工具栏上的“测量描”按钮。 </li><li>选择“显示跟踪测量”选项“NeuronJ：测量”窗口，选择“运行”。 </li><li> “NeuronJ：描”窗口中选择“文件”。 </li><li>选择“另存为”保存文件作为filename_info。 “文件名”必须完全匹配的原始图像文件（包括大小写），由_info的名称，不应该包含3个字母的文件扩展名。例如，对于原始图像名称Cell20.tif，这个文件将被命名为“Cell20_info”。检查您的计算机不会自动添加。xls文件扩展名。如果确实如此，必须手动删除的文件扩展名。 </li></ol></li></ol> 4。使用篝火构建NeuronJ数据的初步SWC文件。 <ol><li>使用“bonfire_load重组文件夹” <ol class="lalpha"><li>双点击图标打开MATLAB。 </li><li>在命令窗口的右上角点击“”按钮。 </li><li>选择“篝火”在您的主文件夹“浏览文件夹”窗口中的文件夹。 </li><li>类型“bonfire_load到MATLAB命令窗口，然后按ENTER键。 </li><li>选择的条件，要分析在“浏览文件夹”窗口，选择“OK”的文件夹。 </li><li>这将重组，通过创建细胞子文件夹包含所有为每个单元格的数据文件夹结构。 </li></ol></li><li>创建使用&#39;bonfire_ndf2swc“的filename_prelim.swc文件<ol class="lalpha"><li>键入“bonfire_ndf2swc”进入命令窗口，然后按ENTER键。 </li><li>选择相同的条件下的文件夹，你只是在“浏览文件夹”窗口“bonfire_load”重组并选择“确定”。 </li><li>这将创建一个在每个单元的文件夹。SWC文件选择条件。现在每个单元文件夹将包含5个文件;原来的TIF图像，NDF文件，_info的跟踪标识符文件，TXT文件和SWC文件。。。 </li></ol></li></ol> 5。使用NeuronStudio敲定SWC文件。 <ol><li>打开和校准NeuronStudio神经元图像<ol class="lalpha"><li>打开NeuronStudio的程序。 </li><li>选择“文件→打开NeuronStudio工具栏上。 </li><li>找到你要编辑并打开它的神经元。TIF图像。 </li><li>选择“运行”→“设置，输入”3（X，Y，Z）在盒“体素大小”窗口“。 </li><li>选择“文件→导入深港西部通道。选择适当的SWC文件。现在的图像文件将被覆盖与跟踪图像。细胞SOMA应覆盖一个红色圆圈。 </li></ol></li><li>使用NeuronStudio链接突起<ol class="lalpha"><li>在NeuronStudio使用的工具，正确识别分支点和结束点（我们建议您成为熟悉分析数据之前NeuronStudio的功能和快捷键）。 </li><li>具体来说，使用的“突起工具”加入节点，以便： <ul><li>每个分支点（黄节点）只能创建两个分支</li><li>所有痕迹连续与SOMA（只有一个红色节点） </li></ul></li></ol></li><li>从NeuronStudio数据导出<ol class="lalpha"><li>选择“文件→保存突起（保存为默认名称）”。 </li></ol></li><li>在错误检查。SWC文件使用“bonfire_trace_check” <ol class="lalpha"><li>键入“bonfire_trace_check到MATHLAB命令窗口，然后按ENTER键。 </li><li>选择条件的文件夹，包含你刚才在“浏览文件夹”窗口中处理的数据，并选择“确定”。 </li><li>如果有任何文件夹中的图像的任何错误，程序将输出图像显示错误的位置是位于。 </li><li>修复错误在NeuronStudio <ol class="lroman"><li>打开图像文件保存在出口一步。 </li><li>找到并解决的问题（S）。 </li><li>单击“文件→保存突起。 </li><li>重复其他图像，需要修复。 </li><li>重新运行“bonfire_trace_check”（5.4）。 </li></ol></li></ol></li></ol> 6。使用“篝火”，从SWC文件中提取形态学数据。： <ol class="lalpha"><li>键入“篝火”进入命令窗口，然后按ENTER键。 </li><li>选择条件文件夹包含您要分析的“浏览文件夹”窗口中的数据，并选择“确定”。 </li><li>篝火分析会生成一个窗口为每个被分析的神经元，它的图形与在分析中使用的肖尔环的神经元的形态。此外，“篝火”命令将生成。mat文件包含所有已采取从分析的形态学信息。 </li></ol> 7。使用“bonfire_results查看数据： <ol class="lalpha"><li>到MATLAB命令窗口中键入“bonfire_results”。 </li><li>选择条件为条件，你想查看“浏览文件夹”窗口，选择“OK”文件夹。 </li><li>的“浏览文件夹”窗口，将暂时关闭并重新打开，让你选择你想观看的附加条件。 </li><li>当您完成选择条件的文件夹，选择“取消”退出遴选过程。 </li><li> “Bonfire_results”将返回摘要图表，包括日您选择的条件文件夹E数据。 </li></ol> 8。使用“bonfire_export数据导出到Excel： <ol class="lalpha"><li>在命令窗口中键入“bonfire_export”。 </li><li>选择条件的文件夹中包含的数据，你想出口在“浏览文件夹”窗口，选择“OK”。 Bonfire_export将创建的形态数据的Excel文件将放置在他们选择的条件文件夹。 </li></ol> 9。代表性的成果： 如图3所示的数据包含两个条件的设置上的篝火方案所产生的数据的一个例子。在这个例子中，条件1神经细胞中含有较多的突起，远端的细胞体。例如图像（ 图3B），以及在总树突状乔木（ 图3A）肖尔曲线，并在图形终端点（ 图 3C），可以观察到这种现象。此外，由于篝火方案还执行肖尔的图像分区域分析，我们能够更具体的确定有增加的突起的身份。 三阶或更大的突起（ 图3F），中介和终端突起总数的交点总数（图 3G）增加远端的细胞体。这些趋势也可以观察到的数字3D和3E。 <img src="/files/ftp_upload/2354/2354fig1.jpg" alt="图1" /> 图1：篝火程序流程图神经元 。使用ImageJ跟踪。然后，将数据的出口，并转换成是初步的。SWC文件的篝火方案。 NeuronStudio是用于定义连接的突起。篝火检查错误，然后计算肖尔曲线，小学，中学，以及更高的秩序突起，分支点和突起的提示。最后，数据导出到Excel进行统计分析。 <img src="/files/ftp_upload/2354/2354fig2.jpg" alt="图2" /> 图2：文件结构所需的篝火分析文件的结构必须符合或程序将无法正常运行 。文件夹和文件，文件夹和文件的数量，名称可以改变。 <img src="/files/ftp_upload/2354/2354fig3.jpg" alt="图3" /> 图3：从篝火方案示例输出数据 a）总肖尔曲线 。二）范例倒了这两个条件的图像。三）分支点和终端点/细胞的平均数量。四）流程/细胞的平均人数为小学，中学，大专或更大的突起。五）流程/根，中间细胞和终端突起的平均人数。 F）的分部特定身份肖尔分析曲线。段分为小学，中学，大专或更大。 G）的分部特定身份肖尔分析曲线。段归根段，中间段，或终端市场。

<ol>
	<li>Elston, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidal+cells+of+the+frontal+lobe:+all+the+more+spinous+to+think+with.">Pyramidal cells of the frontal lobe: all the more spinous to think with.</a> J Neurosci. 20, (2000).</li><li>Koch, C., Segev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+single+neurons+in+information+processing.">The role of single neurons in information processing.</a> Nat Neurosci. , 1171-1177 (2000).</li><li>Poirazi, P., Mel, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+active+dendrites+and+structural+plasticity+on+the+memory+capacity+of+neural+tissue.">Impact of active dendrites and structural plasticity on the memory capacity of neural tissue.</a> Neuron. 29, 779-796 (2001).</li><li>Schaefer, A. T., Larkum, M. E., Sakmann, B., Roth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coincidence+detection+in+pyramidal+neurons+is+tuned+by+their+dendritic+branching+pattern.">Coincidence detection in pyramidal neurons is tuned by their dendritic branching pattern.</a> J Neurophysiol. 89, 3143-3154 (2003).</li><li>Vetter, P., Roth, A., Hausser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+action+potentials+in+dendrites+depends+on+dendritic+morphology.">Propagation of action potentials in dendrites depends on dendritic morphology.</a> J Neurophysiol. 85, 926-937 (2001).</li><li>Hausser, M., Spruston, N., Stuart, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+dynamics+of+dendritic+signaling.">Diversity and dynamics of dendritic signaling.</a> Science. 290, 739-744 (2000).</li><li>Brette, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulation+of+networks+of+spiking+neurons:+a+review+of+tools+and+strategies.">Simulation of networks of spiking neurons: a review of tools and strategies.</a> J Comput Neurosci. 23, 349-398 (2007).</li><li>Arendt, T., Zvegintseva, H. G., Leontovich, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+changes+in+the+basal+nucleus+of+Meynert+and+in+the+diagonal+band+nucleus+in+Alzheimer's+disease--a+quantitative+Golgi+investigation.">Dendritic changes in the basal nucleus of Meynert and in the diagonal band nucleus in Alzheimer's disease--a quantitative Golgi investigation.</a> Neuroscience. 19, 1265-1278 (1986).</li><li>Harrison, P. 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+neuropathology+of+schizophrenia.+A+critical+review+of+the+data+and+their+interpretation.">The neuropathology of schizophrenia. A critical review of the data and their interpretation.</a> Brain. 122, 593-624 (1999).</li><li>Lewis, D. A., Glantz, L. A., Pierri, J. N., Sweet, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cortical+glutamate+neurotransmission+in+schizophrenia:+evidence+from+morphological+studies+of+pyramidal+neurons.">Altered cortical glutamate neurotransmission in schizophrenia: evidence from morphological studies of pyramidal neurons.</a> Ann N Y Acad Sci. 1003, 102-112 (2003).</li><li>Kaufmann, W. E., Moser, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+anomalies+in+disorders+associated+with+mental+retardation.">Dendritic anomalies in disorders associated with mental retardation.</a> Cereb Cortex. 10, 981-991 (2000).</li><li>Georges, P. C., Hadzimichalis, N. M., Sweet, E. S., Firestein, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yin-yang+of+dendrite+morphology:+unity+of+actin+and+microtubules.">The yin-yang of dendrite morphology: unity of actin and microtubules.</a> Mol Neurobiol. 38, 270-284 (2008).</li><li>Firestein, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cypin:+a+cytosolic+regulator+of+PSD-95+postsynaptic+targeting.">Cypin: a cytosolic regulator of PSD-95 postsynaptic targeting.</a> Neuron. 24, 659-672 (1999).</li></ol>

作者宣称没有利益争夺。供资机构在篝火的发展并没有科学的作用。

篝火方案是一个半自动化的方案，枝蔓和轴突形态分析。这大大增加了超过手动执行分析肖尔分析的效率和准确性。此外，篝火程序保存在每一步的过程数据，从而有可能审核数据，并验证了分析的准确性。因此，数据分析的任务可以被分配给许多个人，而不影响精度。最后，由执行上的图像分区域进行分析，该方案是能够识别肖尔分析仅仅是对整个枝蔓或轴突乔木运行时错过当地的分支。我们?...

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<td>NeuronJ plugin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td><a href="http://www.imagescience.org/meijering/software/neuronj/">http://www.imagescience.org/meijering/software/neuronj/</a></td>
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<td>ImageJ software</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td><a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a></td>
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<td>Bonfire program</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td><a href="http://lifesci.rutgers.edu/~firestein">http://lifesci.rutgers.edu/~firestein</a></td>
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<td>NeuronStudio</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td><a href="http://research.mssm.edu/cnic/tools-ns.html">http://research.mssm.edu/cnic/tools-ns.html</a></td>
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<td>MatLab Program</td>
<td>&nbsp;</td>
<td>MathWorks</td>
<td>&nbsp;</td>
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automated sholl analysis of digitized neuronal morphology at multiple scales

神经元的形态起着重要作用，决定神经元功能和沟通 1-3 。具体来说，它会影响神经元接收来自其他细胞的投入能力 2的动作电位 4,5的传播作出贡献。也影响了信息处理的突起的形态。树突形态的多样性，促进本地和远距离信号，并允许单个神经元或神经元组进行6,7神经网络内专门的职能。在枝晶形态的改变，包括树突分支模式的变化的碎片，已经观察到在一些疾病状态，其中包括阿尔茨海默氏病 ，精神分裂症9,10， 精神发育迟滞11。既了解形状枝晶形貌的因素，并确定在枝晶形貌变化的能力是在神经系统的功能和功能障碍的认识至关重要。 突起的形态往往是分析肖尔分析和计算突起和枝梢。这种分析是普遍适用的树突，但它也可以应用到轴突。手工执行这个分析既耗时，不可避免地引入了变异由于实验者的偏见和不一致。篝火方案是一个半自动化的枝蔓和轴突形态时提供开放源码的形态分析工具为基础的分析方法。我们的计划，使当地在枝蔓和轴突分支行为的变化神经炎乔木的分区域执行肖尔分析检测。例如，肖尔分析是神经元树突和轴突的图案是由许多细胞内外因素的影响，在每个进程的一个子集（小学，中学，终端，根等），以及作为一个整体，许多署理本地。因此，由此产生的乔木形态，是一种作用于特定的突起，因此有必要执行规模较小的形态分析， 以观察这12个地方差异的具体进程的结果。 篝火程序需要使用两个开源分析工具，NeuronJ插件ImageJ和NeuronStudio。神经元追踪ImageJ，NeuronStudio是用来定义突起之间的连接。篝火包含在MATLAB（MathWorks公司）编写的自定义脚本是用来作进一步的分析数据转换成相应的格式，请检查用户的错误，并最终执行肖尔分析。最后，数据导出到Excel进行统计分析。一个篝火程序的流程图如图1所示。

Watch this Scientific Journal Video about Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales at JoVE.com

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

我们已经开发出一种计算机程序来分析神经元的形态。在两个现有的开源分析工具的结合，我们的计划执行肖尔，分析和确定的突起，分支点和突起的提示。进行分析，使地方在突起的形态变化可以观察。

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 ...

automated-sholl-analysis-digitized-neuronal-morphology-at-multiple

Research

JoVE Journal

Neuroscience

33.3K 次观看.  Rutgers University. 我们已经开发出一种计算机程序来分析神经元的形态。在两个现有的开源分析工具的结合，我们的计划执行肖尔，分析和确定的突起，分支点和突起的提示。进行分析，使地方在突起的形态变化可以观察。

视频: Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

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 ...

科研

神经科学

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

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 ...

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

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

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

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

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 ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

在脑核糖凝视与原子力显微镜

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

在多发性硬化症的小鼠模型测量进行性神经功能残疾

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

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 ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

慢性压迫性损伤小鼠步态的自动分析

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

嗜德维人神经元树突状树突状复杂度的定量分析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...