，NRSA（F32-NS-066698; DPS）;工作是由史密斯家庭基金会（BS），达纳基金会（BS），约翰·默克学者奖励计划（BS），NINDS（BS RO1-NS-07100801）资助项目，南希·劳瑞商标基金会（DPS），美国国立卫生研究院（P30-HD-18655; MRDDRC影像核心）。

1，顺行研资局突触前输入的标签注意：所有涉及使用动物实验，根据美国国立卫生研究院的所有准则审阅及机构动物护理和使用委员会（IACUC）监督。 <ol><li>消毒领域和手段。 </li><li>麻醉用鼠标在一个树脂玻璃感应室4％（体积）的异氟醚（这个体积％异氟醚适用于新生儿，成年小鼠）。观察小鼠的密切合作，以避免过度麻醉。注意：避免吸入用真空废气撤离。 </li><li>经过1-3分钟，（年纪较大的老鼠将需要更少的时间）确保麻醉的适当水平是通过捏尾巴（无反应应观察），并观察呼吸频率（速度应缓慢而稳定的）实现的。 </li><li>把鼠标在其侧面及地方鼻锥递送3-4％（体积）的异氟烷在炉鼻的立体显微镜下（新生儿&lt;产后10天（P10）〜4％（体积）;&gt; P10〜3％（体积））。 </li><li>露出巩膜。为新生儿注射前的眼图张开，用一双小弹簧剪刀剪开眼睑拉背部皮肤，露出巩膜。对于年纪较大的老鼠，用手指拉回来眼睛周围，露出巩膜皮肤。注意：在新生儿中，有时是在用眼角的余光另一个垂直切割是必要的。割双眼皮的角落里时，因为有一个血管，如果切割，会流血过多照顾。 </li><li>使用无菌30.5号针头刺穿一个小孔眼的一侧在巩膜开始处的线。小心避免通过插入针远远不够的斜面进入眼睛损坏镜头。 </li><li>允许玻璃体流出孔，并使用无菌棉签来吸收液体。 </li><li>一旦玻璃已经停止流淌出来的孔，插入连接到汉密尔顿注射器预装了顺行示踪染料的钝端针入孔。注意：插入针缓慢，以避免刺穿眼睛的另一侧或损坏透镜。 </li><li>慢慢的染料注入到眼睛。通常，霍乱毒素β亚基偶联于Alexa的594，647，或488（CTB-594，CTB-647，或CTB-488，5-6微克/微升）用于顺行迹RGC输入。新生儿（≤10页）1-2微升充足且对小鼠&gt; P10〜3微升就足够了。注：Alexa染料是溶酶体降解特别耐</li><li>留针在洞口几秒钟，然后慢慢放开。 </li><li>用棉棒吸去多余的液体，防止染料外泄。 </li><li>取少量抗生素软膏的眼睛。如果眼睛被打开了手术，轻轻地重新定位眼皮在一起。 </li><li>如果注入双眼，在另一只眼睛重复过程。 </li><li>注射后（次），离开鼠标下的热灯或热垫，直到它开始从anesth恢复ESIA。 </li><li>返回鼠标到一个干净的家笼和监控，以确保它是返回到殖民地之前完全清醒。 </li></ol> 2，准备组织成像这个组织制备协议用 ​​于在其中吞噬细胞标记有荧光标记物（ 例如 ，CX3CR1-EGFP为小胶质细胞）报告小鼠。如果记者行不可用，研究者可免疫染色的组织切片（见讨论）。 <ol><li> 〜24小时，在注射后，牺牲了鼠标和解剖大脑。 </li><li>落固定在一个Falcon管中过夜，在4℃下填充有4％多聚甲醛（PFA）的脑注意：PFA是有毒的。通过使用在通风橱中或在下沉气流表和佩戴个人防护装备，避免吸入和皮肤接触。注：固定可短（≥4小时）。另外，代替降定影，4％PFA灌注可用于后跟一个2-24小时滴定位。然而，灌流的比较d来固定掉落新生儿和成人的大脑发现图像质量或定量没有区别。 </li><li>漂洗在磷酸盐缓冲盐水（PBS）中脑3倍。 <ol><li>倒大脑和PFA到一个空的权衡船。 </li><li>用刮刀给大脑转移到另一个称量舟充满PBS。 </li><li>脑洗两次在PBS（转移大脑两个称量皿充满PBS）。 </li></ol></li><li>脑转移到Falcon管中填充了30％的蔗糖溶液。离开大脑中的蔗糖在4℃直至大脑沉到管（24-48小时）的底部。 </li><li>一旦大脑下沉，准备大脑切片。下面的步骤是准备用一个滑动切片机用冷冻阶段40μm的浮动部（低温恒温器也可以被使用）。 <ol><li>用刀片去掉不需要的大脑的部分（外膝体，消除大脑的头端和尾端部分）。 </li><li>冻结胸罩在铝箔在干冰。在这段时间内，冷冻切片机阶段和填充一个24孔板的每个孔用0.5ml的M 0.1的磷酸盐缓冲液（PB）。 </li></ol></li><li>装载冷冻大脑（它应该会出现不透明）在冻结阶段。 <ol><li>取少量的最佳切削温度化合物（OCT）的舞台。 </li><li>一旦华侨城开始冻结，奠定了大脑中的十月将被削减的大脑到底应朝上（ 例如 ，为外膝体，面对尾朝上）。 </li><li>覆盖大脑和OCT具有非常精细粉碎的干冰。 </li><li>离开干冰上脑/华侨城〜30秒。 </li><li>使用大画笔，除去干冰。大脑和OCT应冻结舞台。 </li></ol></li><li>首先通过组织切片，直到感兴趣区域（ROI）的区域为止。 </li><li>一旦投资回报率达到，用小画笔从刀片取出部分，并将它们转移到24孔平台Ë含0.1M PB（步骤2.5.2）。保持油漆刷湿润事先从刀片取出部分润湿它在0.1M的PB。注：部分可留在0.1M PB一夜之间在4°C。 </li><li>一旦部分已收集，可视化的顺行标记荧光解剖显微镜下，选择包含投资回报率的部分。 </li><li>安装在一个用小画笔滑动部分。 <ol><li>适用0.1M的PB一个小型游泳池的充电显微镜幻灯片。 </li><li>转移组织切片在PB的游泳池。 </li><li>用PB和画笔的方向和传播组织。 </li><li>使用的Kimwipe灯芯多余的PB。注意避免排汗关闭部分。 </li><li>允许完全风干。 </li><li>取一小颗的安装介质的每个部分，并安装盖玻片上的顶部（​​22×50毫米，1.5号）。 </li><li>密封滑动的边缘用指甲油和储存在-20°C，直到成像会议。注意：虽然成像在一周的制备忠告，幻灯片可以保持在-20℃下持续数周前成像。 </li></ol></li></ol> 3，组织成像所有图像都在旋转盘共聚焦显微镜（的Ultraview Vox的旋转盘共聚焦显微镜配备二极管激光器（405纳米，445纳米，488纳米，514纳米，561纳米和640纳米））所收购。影像也可以收购任何显微镜获取高分辨率的z栈的能力（ 例如 ，激光扫描共聚焦显微镜，显微镜啶其次是反褶积等）。帧大小通常为1,000×1,000像素。 <ol><li>寻找投资回报率在10倍放大倍率和获取图像。在retinogeniculate系统成像的小胶质细胞，投资回报率是最内侧的外膝体部分。 </li><li>转移到更高的放大倍率（一60X计划阿婆目标，NA = 1.4，通常使用）。 </li><li>收购视图包含使用0.2微米的z一步吞噬细胞第一场秒。 </li><li>获得含15-19每只动物吞噬细胞更多的领域。 </li></ol> 4，准备定量（ImageJ的）图片<ol><li>打开Z堆栈中的ImageJ。 </li><li>转到图像菜单中，选择颜色和分离通道。 </li><li>从含有吞噬物质的通道（s）减去背景。 <ol><li>选择包含吞没材料（ 例如 ，突触前输入）通道窗口。 </li><li>进入处理菜单，并选择减去背景。使用10像素的滚动球半径为于外膝体突触前投入顺行追踪。注：滚球半径凭经验确定。然而，一般应大到不属于背景的部分图像中的最大对象的半径。 </li></ol></li><li>平滑含有吞噬细胞的通道。 <ol><li>选择含有吞噬细胞（ 例如 ，小胶质细胞）的通道窗口。 </li><li>进入处理菜单和SELECT过滤器，平均。使用1.5均值滤波（平滑这在后面的步骤中面绘制的图像）。 </li></ol></li><li>从含有吞噬细胞的通道中减去背景。 <ol><li>选择含有吞噬细胞（ 例如 ，小胶质细胞）的通道窗口。 </li><li>进入处理菜单中，选择减去背景（设置将需要凭经验确定）。使用50像素的EGFP阳性的小胶质细胞滚动球半径。 </li></ol></li><li>通过进入图片菜单，然后选择颜色，合并通道合并通道。 </li><li>作物的ROI含吞噬细胞从合并后的文件（这使得表面渲染速度更快，请参阅步骤5）。 <ol><li>使用绘制矩形选择工具，在工具栏中围绕投​​资回报率一箱。 </li><li>转到图像菜单，然后选择裁剪。 </li></ol></li><li>再次分裂通道（见步骤4.2）。 </li><li>每个通道保存为自己的TIFF文件。 </li></ol> 5，准备用于图像定量在Imaris里<ol><li>打开Imaris里，并确保音量图标在左上角的菜单（ 图2）中选择。 </li><li>裁剪图像的打开第一通道（任何频道都可以先开，只是保持一致）。 </li><li>添加其他通道（s）。进入编辑菜单，选择添加通道和选择文件中的下一个频道。 （重复任何剩余的渠道这一步）。注：要更改颜色，请进入显示调整窗口，点击频道名称（ 图3A）。这将弹出一个对话框来调整颜色。如果显示调整窗口不可见，请进入编辑菜单，然后选择显示调整。 </li><li>调整像素值。进入编辑菜单，然后选择图像属性。在这个窗口中，调整的x，y和z的像素值（这些值取决于显微镜，物镜，摄像头，和z步收购，可以在用于获取图像的软件中找到）。 </li><li>步骤5.5将导致一个非常小图像。要调整，请按一下右下角（ 图2）的拟合图标。 </li><li>在显示调整窗口，调整亮度和对比度，使用左侧和中间的三角形的每个信道。 </li></ol> 6，3维（3D）表面的吞噬细胞的渲染<ol><li>在顶部的工具栏，可以肯定的超越模式的选择（ 图3C）。 </li><li>在显示调整窗口中，取消选中除吞噬细胞通道的所有通道。这会从视野中移除它们。 </li><li>点击面绘制图标（ 图2）。 </li><li>一个创建的窗口将显示在左下角（ 图4A），确保分部的投资回报率是选中在这第一个窗口。点击底部的蓝色前进按钮。 </li><li>调整平滑。 <ol><li>在下一个窗口中，从下拉菜单中（ 图4B）吞噬细胞的通道。这将调整细节量将浮出水面并且应根据经验确定（0.1μm的平滑通常用于小胶质细胞）。 </li><li>选择绝对阈值。 </li><li>点击蓝色的前进按钮在底部。 </li></ol></li><li>阈值的图像。 <ol><li>点击直方图并拖动，直到图像被适当地浮出水面（ 图4C）。这是通过旋转该图像，以确保覆盖在荧光图像上的灰度表面是一个精确的表面表示经验确定。注：要旋转，在指针菜单中选择浏览在上屏幕右侧（ 图3B）中，单击并按住图像旋转。要恢复图像至原来的方向，选择地底从视图菜单中左。 </li><li>点击蓝色的前进按钮在底部。 </li></ol></li><li>在接下来的窗口中，有一个选项按大小等。除非图像是非常嘈杂的过滤掉任何表面，不过滤和删除Ÿ过滤器，自动出现。点击绿色箭头在底部完成面绘制的吞噬细胞。一组新的选项卡会出现（ 图5） </li><li>如有必要，删除不感兴趣的吞噬细胞的一部分的任何表面。 <ol><li>确保在指针菜单（ 图3B）选中选择选项。 </li><li>单击要删除的任何表面，使它变成黄色。若要删除多个曲面，单击表面上，同时按住控制钮。 </li><li>点击铅笔选项卡下删除。 </li></ol></li><li>选择和合并的吞噬细胞的所有表面形成一个表面上。 <ol><li>点击漏斗（ 即过滤器选项卡， 图5C）。 </li><li>单击添加。 </li><li>选择任何过滤器，直方图拖动到最左边（所有表面应为黄色）。 </li><li>回到铅笔选项卡并单击统一。 </li></ol></li><li>进入图形选项卡（ 图5A </sTRONG&gt;），然后单击详细信息，并选择任何参数来显示。选择卷（ 图5D）。对于显示多个参数，去左下角（ 图2）的扳手图标，然后选择参数进行记录。 </li><li>记录吞噬细胞的体积和文件，然后再继续保存。 </li></ol>吞噬材料7。三维表面渲染<ol><li>创建已吞噬细胞引起的吞噬物质的新的信道。 <ol><li>而吞噬细胞表面被选中，单击铅笔标签。 </li><li>单击面膜所有。 </li><li>在出现的对话框中，选择相应的吞噬材料（ 例如 ，突触前投入顺行追踪; 图5B）的通道。 </li><li>检查是否重复申请通道面膜前，点击OK。 </li><li>一个新的通道会出现在一个只包含已吞噬的物质和里面的显示调整窗口吞噬细胞。 </li></ol></li><li>表面渲染含总吞噬和nonengulfed材料的通道。 <ol><li>取消选中在显示调整窗口的所有通道，除了要浮出水面的通道，并取消对吞噬细胞产生的表面。 </li><li>再次点击面绘制图标（见步骤6.3），并利用所描述的吞噬细胞相同的步骤通道创建一个面（步骤6.4-6.11）。注意事项：一定要选择正确的通道前，平滑和阈值（步骤6.5; 图4B）。记下阈值（见步骤6.6; 图4C）。该值也可以在表面渲染后得到完成（魔杖标签; 图5A）。 </li></ol></li><li>记录总的吞噬和非吞噬物质的体积和文件，然后再继续保存。 </li><li>表面渲染吞噬物质（吞噬细胞内的物质）。 <ol><li>可视化的荧光只吞噬物质的新渠道（见步骤7.2.1）。 </li><li>按照上述相同的步骤（步骤7.2-7.3）。注意事项：一定要选择从下拉菜单中选择正确通道平滑之前（选择步骤7.1中创建新的通道）。在阈值窗口中，手动输入被用于渲染的总吞噬和非吞噬材料（见步骤7.2.2）中设定的阈值。 </li></ol></li><li>记录吞噬物质的量和持续之前保存的文件。 </li></ol> 8，计算视场的总容积<ol><li>任何通道创建一个新的表面。 </li><li>在阈值窗口（步骤6.6），滑动直方图一路向左，使整个场浮出水面。 </li><li>堆焊完成整场并记录量（步骤6.7-6.11）。 </li><li>保存该文件。 </li></ol> 9，计算吞噬物质的量<ol><li>计算的密度使用以下算法总吞噬和非吞噬物质的通道： 场（第8步）的总吞噬和非吞噬材料体积（步骤7.3）/音量。 </li><li>使用下面的算法计算出每个单元％吞没： （吞噬物质的量（步骤7.5）/的吞噬细胞（步骤6.11）的总体积）×100 注意：为了计算变化细胞大小，数据被归一化到吞噬细胞的总体积。如果从步骤9.1数字是高度可变的对面域，它可能需要从步骤9.2中的数据归一化，以计算在9.1。 </li></ol>

<ol>
	<li>Ransohoff, R. M., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+physiology:+unique+stimuli,+specialized+responses.">Microglial physiology: unique stimuli, specialized responses.</a> Annu Rev Immunol. 27, 119-145 (2009).</li><li>Aguzzi, 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=Microglia:+scapegoat,+saboteur,+or+something+else.">Microglia: scapegoat, saboteur, or something else.</a> Science. 339 (6116), 156-161 (2013).</li><li>Tremblay, M. E., 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=The+role+of+microglia+in+the+healthy+brain.">The role of microglia in the healthy brain.</a> J Neurosci. 31 (45), 16064-16069 (2011).</li><li>Hanisch, U. K., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+active+sensor+and+versatile+effector+cells+in+the+normal+and+pathologic+brain.">Microglia: active sensor and versatile effector cells in the normal and pathologic brain.</a> Nat. Neurosci. 10 (11), 1387-1394 (2007).</li><li>Sierra, 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=Janus-faced+microglia:+beneficial+and+detrimental+consequences+of+microglial+phagocytosis.">Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis.</a> Front Cell Neurosci. 7, (2013).</li><li>Dheen, S. T., 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=Microglial+activation+and+its+implications+in+the+brain+diseases.">Microglial activation and its implications in the brain diseases.</a> Curr Med Chem. 14 (11), 1189-1197 (2007).</li><li>Schafer, D. 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=Microglia+sculpt+postnatal+neural+circuits+in+an+activity+and+complement-dependent+manner.">Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.</a> Neuron. 74 (4), 691-705 (2012).</li><li>Katz, L., Shatz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+and+the+constuction+of+cortical+circuits.">Synaptic activity and the constuction of cortical circuits.</a> Science. 274 (5290), 1133-1138 (1996).</li><li>Sanes, J. R., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+vertebrate+neuromuscular+junction.">Development of the vertebrate neuromuscular junction.</a> Annu Rev Neurosci. 22, 389-442 (1999).</li><li>Hua, J. Y., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+and+the+dynamics+of+central+nervous+system+development.">Neural activity and the dynamics of central nervous system development.</a> Nat Neurosci. 7 (4), 327-332 (2004).</li><li>Holtmaat, A., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+structural+synaptic+plasticity+in+the+mammalian+brain.">Experience-dependent structural synaptic plasticity in the mammalian brain.</a> Nat Rev Neurosci. 10 (9), 647-658 (2009).</li><li>Butz, M., 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=Activity-dependent+structural+plasticity.">Activity-dependent structural plasticity.</a> Brain Res Rev. 60 (2), 287-305 (2009).</li><li>Bruel-Jungerman, E., 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=Brain+plasticity+mechanisms+and+memory:+a+party+of+four.">Brain plasticity mechanisms and memory: a party of four.</a> Neuroscientist. 13 (5), 492-505 (2007).</li><li>Schafer, D. P., Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+elimination+during+development+and+disease:+immune+molecules+take+centre+stage.">Synapse elimination during development and disease: immune molecules take centre stage.</a> Biochem Soc Trans. 38 (2), 476-481 (2010).</li><li>Alexander, 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=The+complement+system:+an+unexpected+role+in+synaptic+pruning+during+development+and+disease.">The complement system: an unexpected role in synaptic pruning during development and disease.</a> Annu Rev Neurosci. 35, 369-389 (2012).</li><li>Brown, A., Weaver, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dark+side+of+neuroplasticity.">The dark side of neuroplasticity.</a> Exp Neurol. 235 (1), 133-141 (2012).</li><li>Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+27:+Neural+plasticity+after+nerve+injury+and+regeneration.">Chapter 27: Neural plasticity after nerve injury and regeneration.</a> Int Rev Neurobiol. 87, 483-505 (2009).</li><li>Tan, A. M., Waxman, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury,+dendritic+spine+remodeling,+and+spinal+memory+mechanisms.">Spinal cord injury, dendritic spine remodeling, and spinal memory mechanisms.</a> Exp Neurol. 235 (1), 142-151 (2012).</li><li>Wolpaw, J. R., Tennissen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+spinal+cord+plasticity+in+health+and+disease.">Activity-dependent spinal cord plasticity in health and disease.</a> Annu Rev Neurosci. 24, 807-843 (2001).</li><li>Schafer, D. 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=The+&amp;#34;quad-partite&amp;#34;+synapse:+Microglia-synapse+interactions+in+the+developing+and+mature+CNS.">The &amp;#34;quad-partite&amp;#34; synapse: Microglia-synapse interactions in the developing and mature CNS.</a> Glia. 61 (1), 24-36 (2013).</li><li>Mukhopadhyay, S., 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=Manganese-induced+trafficking+and+turnover+of+the+cis-Golgi+glycoprotein+GPP130.">Manganese-induced trafficking and turnover of the cis-Golgi glycoprotein GPP130.</a> Mol Biol Cell. 21 (7), 1282-1292 (2010).</li><li>Jung, S., 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20, 4106-4114 (2000).</li><li>Knobloch, M., Mansuy, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spine+loss+and+synaptic+alterations+in+Alzheimer's+disease.">Dendritic spine loss and synaptic alterations in Alzheimer's disease.</a> Mol Neurobiol. 37 (1), 73-82 (2008).</li><li>Masliah, E., 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=Synaptic+remodeling+during+aging+and+in+Alzheimer's+disease.">Synaptic remodeling during aging and in Alzheimer's disease.</a> J Alzheimers Dis. 9 (3 Suppl), 91-99 (2006).</li><li>Yoshiyama, 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=Synapse+loss+and+microglial+activation+precede+tangles+in+a+P301S+tauopathy+mouse+model.">Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model.</a> Neuron. 53 (3), 337-351 (1016).</li><li>Milnerwood, A. J., Raymond, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+synaptic+pathophysiology+in+neurodegeneration:+insights+from+Huntington's+disease.">Early synaptic pathophysiology in neurodegeneration: insights from Huntington's disease.</a> Trends Neurosci. 33 (11), 513-523 (2010).</li><li>Noda, M., Suzumura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sweepers+in+the+CNS:+Microglial+Migration+and+Phagocytosis+in+the+Alzheimer+Disease+Pathogenesis.">Sweepers in the CNS: Microglial Migration and Phagocytosis in the Alzheimer Disease Pathogenesis.</a> Int J Alzheimers Dis. , (2012).</li><li>Rotshenker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+macrophage+activation+and+the+regulation+of+complement-receptor-3+(CR3/MAC-1)-mediated+myelin+phagocytosis+in+injury+and+disease.">Microglia and macrophage activation and the regulation of complement-receptor-3 (CR3/MAC-1)-mediated myelin phagocytosis in injury and disease.</a> J Mol Neurosci. 21 (1), 65-72 (2003).</li><li>Smith, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+myelin+in+demyelinative+disease:+a+review.">Phagocytosis of myelin in demyelinative disease: a review.</a> Neurochem Res. 24 (2), 261-268 (1999).</li></ol>

没有利益冲突的声明。

为了准确地测量吞噬作用，吞噬材料必须贴在这样一种方式，研究人员可以直观一次溶酶体降解发生。此外，高分辨率成像是必需的，随后使用软件，使研究者能够可视化整个细胞的体积和量化其内容。在这个协议中，我们描述了用于使用CTB偶联至Alexa染料来标记吞噬材料结合高分辨率共焦显微镜和三维重建测定吞噬细胞介导的​​吞噬一个高度可靠的和定量的方法。这种方法已被用来在实验室?...

<html>突触电路改造整个动物的生命。在脑发育，突触形成过剩，而且必须经过突触的修剪涉及选择性去除突触的一个子集和那些仍然8-10突触的维持和加强。这个过程是必需的，以实现对成人神经系统的精确的连接特性。在成人中，突触也可以是塑料，特别是在学习和记忆的上下文中。此可塑性的结构相互关系被认为是包括树突棘和突触前boutons 11-13的加法和/或消除。除了 ​​在健康的神经系统这些角色，突触重塑也参与了神经系统疾病/伤害12,14,15。例如，下面的脊髓损伤，轴突切断后必须改造，形成新的突触，实现功能的恢复16-19。 NT“&gt;新兴突触可塑性的一个重要方面是吞噬或拟用于去除3,5,20突触吞噬的过程中，我们最近发现这种现象在健康，出生后小鼠大脑突触7修剪的情况下，特别，小胶质细胞，常住中枢神经系统的免疫细胞和吞噬细胞，被证明在高峰期和发育突触的修剪，丘脑的产后背外侧膝状体核（外膝体）的区域吞没突触前输入。这吞没的遗传或药理封锁导致突触连接持续的赤字。 在这个协议中，我们描述了一种可靠的和高度的定量测定法来测量的突触前输入，吞噬细胞介导的​​吞噬。对于这篇文章的目的，此法将在发展中retinogeniculate系统，其中包括视网膜神经节细胞（RGC的）居住在视网膜的背景下提出的项目突触前输入到外膝体（ 图1A）。开始，溶酶体降解抗性顺行标记策略进行说明，这是用于可视化的外膝体RGC特异突触前输入（ 图1）7,21。以下本说明书中，用于成像的详细方法和使用共聚焦显微镜并结合3维（3D）表面体绘制的定量测定吞噬将得到。此方法是基于固定的组织制备，但也可以适于在实时成像研究使用。重要的是，尽管已经测定在健康，产后retinogeniculate系统的上下文中得到证实，人们可以应用相同的技术来评估其它吞噬细胞 - 神经元相互作用的整个大脑和疾病过程中，以及在其他器官系统的吞噬细胞的功能。

<table border="0" cellpadding="0" cellspacing="0" width="533">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:144px;">Heat pad</td>
			<td style="width:159px;">Vet Equip, Inc.</td>
			<td style="width:168px;">965500&#160;</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:144px;">Warm water source for heat pad</td>
			<td style="width:159px;">Kent Scientific</td>
			<td style="width:168px;">TP-700</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:144px;">Stereo microscope</td>
			<td style="width:159px;">DSC Optical</td>
			<td style="width:168px;">Zeiss Opmi -6 Surgical Microscope</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="40" rowspan="2" style="height:40px;width:144px;">Sliding microtome with freezing stage</td>
			<td rowspan="2" style="width:159px;">Leica</td>
			<td rowspan="2" style="width:168px;">SM2010 R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:144px;">Microtome blade</td>
			<td style="width:159px;">Leica</td>
			<td style="width:168px;">14021607100</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:144px;">Fluorescent dissecting microscope</td>
			<td style="width:159px;">Nikon</td>
			<td style="width:168px;">SMZ800 with Epi-fluorescence attachment</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:144px;">Spinning disk confocal microscope</td>
			<td style="width:159px;">Perkin Elmer</td>
			<td style="width:168px;">UltraView Vox Spinning Disk Confocal</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">10 &#181;l Hamilton gas tight syringes</td>
			<td style="width:131px;">Hamilton</td>
			<td style="width:116px;">80030</td>
			<td style="width:144px;">Use a different syringe for each color dye/tracer</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Hamilton needles</td>
			<td style="width:131px;">Hamilton</td>
			<td style="width:116px;">7803-05, specifications: blunt, 1.5&#34;</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="20">
			<td height="54" rowspan="3" style="height:54px;width:131px;">Alexa-conjugated cholera toxin &#946; subunit (CTB)</td>
			<td rowspan="3" style="width:131px;">Invitrogen</td>
			<td style="width:116px;">488: C22841</td>
			<td rowspan="3" style="width:144px;">Reconstitute in sterile saline, 80 &#181;l (488), 100 &#181;l (594), 20 &#181;l (647)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:116px;">594: C22842</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:116px;">647: C34778</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Phosphate Buffered Saline&#160;(PBS)</td>
			<td style="width:131px;">Sigma</td>
			<td>P4417-50TAB</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:131px;">Neomycin and Polymyxin B Sulfates and Bacitracin Zinc Ophthalmic Ointment USP (antibiotic ointment)</td>
			<td style="width:131px;">Bausch &#38; Lomb</td>
			<td style="width:116px;">24208-780-55</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">30.5 gauge needle</td>
			<td style="width:131px;">Becton Dickinson</td>
			<td style="width:116px;">305106</td>
			<td rowspan="3" style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Spring scissors</td>
			<td style="width:131px;">Roboz</td>
			<td style="width:116px;">RS-5630</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Cotton-tipped applicator</td>
			<td style="width:131px;">Fisher</td>
			<td style="width:116px;">23-400-125</td>
		</tr>
		<tr height="21">
			<td height="104" rowspan="2" style="height:104px;width:131px;">Paraformaldeyde (PFA)</td>
			<td rowspan="2" style="width:131px;">Electron Microscopy Sciences</td>
			<td rowspan="2" style="width:116px;">15710</td>
			<td rowspan="2" style="width:144px;">Dilute 16%to 4% in PBS. Paraformaldehye is toxic, use&#160; in a fume hood and wear personal protective equipment.</td>
		</tr>
		<tr height="83">
		</tr>
		<tr height="36">
			<td height="138" rowspan="4" style="height:138px;width:131px;">Dissection tools &#8211; scissors, forceps, spatula</td>
			<td style="width:131px;">Small scissors: Fine Science Tools</td>
			<td style="width:116px;">Small scissors:14370-22</td>
			<td rowspan="4" style="width:144px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Large scissors: Roboz</td>
			<td style="width:116px;">Large scissors: RS-6820</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">#55 forceps: Fine Science Tools</td>
			<td style="width:116px;">#55 forceps: 11255-20</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Spatula: Ted Pella, Inc.</td>
			<td style="width:116px;">Spatula: 13504</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:131px;">Sucrose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:116px;">S8501-5KG</td>
			<td style="width:144px;">Make 30% sucrose in PBS (weight/vol)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">OCT Compound</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">25608-930</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Weigh boat</td>
			<td style="width:131px;">USA Scientific</td>
			<td style="width:116px;">2347-1426</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">24-well plates</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:116px;">353047</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Sodium phosphate monobasic</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:116px;">S6566-500G</td>
			<td rowspan="2" style="width:144px;">Make 0.2 M sodium phosphate monobasic (PB-A) in ddH20 and 0.2 M sodium phosphate dibasic (PB-B) in ddH20.&#160; To make 0.1 M PB, combine 19 ml PB-A and 81 ml PB-B, fill to 200 ml with ddH20&#160;&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:131px;">Sodium phosphate dibasic&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td>S5136-500G</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Coverslips, 22 X 50 mm, No. 1.5</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">48393 194</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Charged microscope slide</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">48311-703</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Vectasheild</td>
			<td style="width:131px;">Vector Laboratories</td>
			<td style="width:116px;">H-1200</td>
			<td style="width:144px;"></td>
		</tr>
	</tbody>
</table>

an engulfment assay: a protocol to assess interactions between cns phagocytes and neurons

吞噬作用是一个过程，其中一个细胞吞噬材料（整个小区的小区，杂物等部件）在其周围的细胞外环境，并随后消化此材料，通常通过溶酶体降解。小胶质细胞是中枢神经系统（CNS）的细胞的吞噬功能的广泛的神经退行性疾病（ 例如 ，β-淀粉样蛋白的间隙在阿尔茨海默氏病）的健康脑的发展（ 例如，突触条件已经描述的驻地免疫细胞修剪）1-6。下面的协议是制定可视化和量化，在发育中的小鼠retinogeniculate系统7的突触前输入，小神经胶质细胞介导的吞噬的吞噬测定。虽然这种测定法用于评估在此特定情况下的小胶质细胞的功能，类似的方法可用于评估其他吞噬细胞在整个脑（ 例如，星形胶质细胞）和身体的其余部分（ 例如 ，外周血巨噬细胞）以及在其中突触重塑发生（ 例如 ，脑损伤/疾病）其他上下文中。

最近，我们使用这个吞噬试验，以可视化和量化的显影retinogeniculate系统的突触前输入，小神经胶质细胞介导的吞噬（ 图1）7。从CX3CR1-EGFP杂合子小鼠视网膜神经节细胞进行顺行追踪与CTB-594和CTB-647进入左眼和右眼分别。在此之后追踪，外膝体中EGFP阳性的小胶质细胞进行成像。这些图像随后的表面呈现为体积测量。 使用这种技术，我们发现，在外膝体（P5?...

Watch this Scientific Journal Video about An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons at JoVE.com

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

小胶质细胞是中枢神经系统（CNS）具有高容量，以在它们的胞外环境吞噬或吞噬物质的驻留的免疫细胞。这里，用于可视化和测量突触小部件的小神经胶质细胞介导的​​吞噬一个广泛适用的，可靠的和高度的定量测定法进行说明。

一个吞噬含量：一个协议，以评估之间的中枢神经系统吞噬细胞和神经元的相互作用

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

an-engulfment-assay-protocol-to-assess-interactions-between-cns

Research

JoVE Journal

Neuroscience

18.1K 次观看.  Boston Children's Hospital, Harvard Medical School. 小胶质细胞是中枢神经系统（CNS）具有高容量，以在它们的胞外环境吞噬或吞噬物质的驻留的免疫细胞。这里，用于可视化和测量突触小部件的小神经胶质细胞介导的​​吞噬一个广泛适用的，可靠的和高度的定量测定法进行说明。 

视频: An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

科研

神经科学

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

幼虫和成人测绘和增强子陷阱Flippase表达中的应用果蝇中枢神经系统

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

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 ...

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

剪切载荷：一个有用​​的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

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 ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

一种方法来提高背根神经节的对齐和髓鞘

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

条纹分析来研究蛋白质底物使用游离海马神经元的吸引力或令人反感的活动

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

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.

间接神经元的星形胶质细胞共培养试验：一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

评估大鼠急性中风后脑恢复的临床前模型

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...