我们要感谢来自威斯康星大学麦迪逊分校电子显微镜设施电子显微镜的大学BK八月。这项工作是由NIH资助R01 - DA026067和P30 - HD03352（JSM）的支持。

1。准备工作1-4 <ol><li>准备一个2.5米的蔗糖浆混合50毫升，500毫升（GM），梯度介质的缓冲，2.5毫升的一个1M的Tris -盐酸，PH7.5股票，0.1毫升0.5 M EDTA，pH值8.0股毫Q水体积。过滤消毒的解决方案，分装和储存冷冻。 </li><li>准备的河豚毒素（TTX）的1000倍的股票，由1毫米的解决方案中的 Milli - Q H 2 O TTX的等分可以储存在-20 ° C </li><li>准备由Milli - Q水解决了100毫米的Tris（pH值7.5），5毫米的Na 2 HPO 4，4毫米的KH 2 PO 4， 碳酸氢钠 3 40毫米，800毫米氯化钠10X刺激缓冲区。股票可以储存于4 ° C。 </li><li>预冷离心机到4 ° C。 </li><li>股票的等渗Percoll（SIP）解决方案，并准备加入Percoll（18毫升）的9卷1体积为2.5米的蔗糖（2毫升）在Milli - Q水拌匀。 </li><li> SIP的款额加入到通用汽车缓冲和混合每个准备3，10，15和23％的梯度层: </li></ol><img alt="表0" src="/files/ftp_upload/3196/3196table0.jpg" /><ol start="7"><li>吹打成贝克曼离心管中倒入2毫升，23，15，10和3％的等渗Percoll解决方案使用一个P1000的吉尔森Pipetman帽梯度层。层与层之间的接口应该清楚没有可辨别的混合层。保存在4 ° C，使用前至少20分钟的梯度。梯度可以存储长达24小时，虽然同一天，建议使用。 [没有经验的实验室人员可能加入蓝色染料isomotic Percoll层界面的可视化解决方案的做法准备梯度。] </li></ol> 2。鼠标清扫1 <ol><li>确保所有的鼠标畜牧业和安乐死的程序是按照美国国立卫生研究院和大学准则执行。安乐死的二氧化碳窒息或批准在动物协议的一种方法的幼崽（P13到P21的年龄）。 </li><li>引脚上的鼠标到夹层板和喷涂的颈部和头部用乙醇的背面。通过脊髓削减颅底一个锋利的剪刀，去除皮肤的头骨顶部，横向切割之间的顶骨和interparietal骨之间的大脑和皮层区域的头骨。然后切基地鼻子沿矢状缝合头骨，小心地取出每个半球侧拉软顶骨，并用抹刀删除皮层。插入大脑小脑以上锅铲，然后舀出皮质，并放置在冰冷的通用缓冲区。立即着手同质化的步骤。不要让皮质坐很长时间在冰冷的缓冲区，因为这将造成不利影响形成的SNS。 </li></ol> 3。制备匀浆和SNS 1-4 <ol><li>在冰冷的通用缓冲冲洗皮质和两个皮层转移到玻璃Dounce匀浆含有5毫升冷通用缓冲区。 </li><li>轻轻同质化的松散杵5-10招皮层（杵的"A"），其次是5-10招紧杵（杵的"B"）。招应迅速，但足够慢，不引起气泡。一个匀浆同质化后的图片是在计划 1 。笔画数会有所不同取决于个人匀浆和随行杵。当完成的梯度应给人以强烈的的SN频段，如果不是，调整笔画数。 </li><li>匀浆转移到15 mL锥形1000xg离心10分钟，在4 °在水平转头C（Allegra的6KR离心机）颗粒细胞碎片和核。纺匀浆的照片是在计划 1 。 </li><li>第2层毫升上清液，每Percoll蔗糖梯度，第管，并在32,500 XG离心5分钟，4 °彗星在一个固定角度转子（美国贝克曼离心机J2 - 21，JA - 17转子）使用适当的适配器（ 试剂和仪器表 ）。 </li><li>移液器起飞和丢弃以上SN频段使用玻璃巴斯德吸管的解决方案。移液器关闭SN频段在15/23％接口，转移到一个圆锥形的冰商店。每个梯度之一皮质会产生0.9-1.1毫升锡。 </li><li>调整的SNS盐的浓度，加入十分之一量的10倍刺激缓冲区，增加1000倍氯化钙 2至终浓度为12纳米，1毫米TTX的股票添加终浓度为1μm，抑制非特异性激发。 （ 氯化钙除了是可选的，如果它会干扰与下游SN应用） 。 </li><li>使用的Micro BCA蛋白检测试剂盒，确定了SNS的蛋白浓度。 （Bradford蛋白测定是不建议，因为从Percoll推断。） </li><li> SNS可以用来直接和迅速的蛋白质translaTION研究。对于其他应用程序的SN裂解可能被清理或集中使用皮尔斯的SDS - PAGE样品制备试剂盒。 </li></ol> 4。代表性的成果: 当鼠标皮质匀浆，并在不连续Percoll蔗糖梯度（ 图1），6条带或分数分离被发现（ 图 1 ）。这是以前对大鼠大脑皮层3,5所示的分子量高频段的成分被破坏细胞膜和髓磷脂和颗粒材料中所含的线粒体或细胞器（ 见表1） 。 23％/ 15％的接口（乐队5）含有丰富的SNS。 如前所述，通过电子显微镜（EM）的乐队在23％/ 15％接口包含完整的SNS，删除和检查。2,6电磁显示存在完整的突触小泡与突触前和突触后成分的保存 （图2）。前和突触后车厢分离是很重要的研究信令和蛋白质的合成，其中发生在两个车厢。7-13 Percoll蔗糖梯度是一个粗略的净化，使我们看到在SN分数轻微的细胞，核和细胞器的污染，但整体分离是好的。 用Western blot检测时，Percoll分数包含在SN带（ 图3，表2）增加突触纯度与预期的蛋白标志物。丰富的SN频段（第五级）均保持在突触和神经元的标记，但其他标志物的丰度显着降低。特别是，GFAP的胶质细胞来源的星形胶质细胞和肿瘤细胞的标记，核纤层蛋白B1，核标记，存在几乎可以忽略不计，而维持或加强的结构和突触标记。此外，还有被保留，在蛋白质合成中发挥的作用的细胞器，如线粒体和高尔基的标记。 典型的产量分别为SN频段250-500纳克每微升的蛋白质，BCA蛋白检测确定。 从头蛋白质合成量的确定活动的SNS，目前由[35S]蛋氨酸掺入（ 图 4）作为监测 。2基底翻译活动增加了1.56倍时，SNS与50微米glutamate/10 μM甘氨酸刺激（GLU）或5.12倍的PIN1抑制剂胡桃启动时。 PIN1的抑制作用，增加蛋白质合成。结果证实，[35S]蛋氨酸掺入的增加是由于从头蛋白质的合成，我们增加了40微米茴香霉素，蛋白质合成抑制剂，并看到了在法团的显着下降在基础水平相比，谷氨酸的存在和缺乏。此外，基底翻译时增加2％TRITON - X 100，这破坏了诚信的SNS，下降了75％。此外，当研究表明最突触标记富集乐队（B4 - B6）， SNS或乐队5 从头蛋白质的合成（ 图5）最大的活动。因此，蔗糖不连续Percoll梯度迅速产生一种非常活跃，丰富SN频段，可用于研究蛋白质翻译。 不连续Percoll蔗糖梯度程序比其他方法更有利，因为苛刻的条件所造成的机械损伤。相比的过滤方法，在清除皮质匀浆，是通过一系列过滤器的大小减少，3,14-15 Percoll方法形成了更大的活动更多的SNS。在图6中可以看出，当超过Percoll蔗糖梯度，有远不如SNS在15/23％接口和更大量的碎膜过滤法编制的SNS等分通过。当从头蛋白质合成是通过S35 -会见纳入计量，过滤法编写的SNS远不如积极（ 图7）。为了最大限度地提高翻译活动，我们利用小鼠P14和P18之间。 SN可以从成年小鼠的准备，但我们观察到的显着增加与少年小鼠（ 图8）编写的SN翻译活动。 <img alt="表1" src="/files/ftp_upload/3196/3196table1.jpg" /> 表1。 3,5 匀浆鼠标皮质不连续Percoll梯度分馏带的成分 。 <img alt="表2" src="/files/ftp_upload/3196/3196table2.jpg" /> 表2。每个乐队的分数相对西方螺栓不连续Percoll蔗糖梯度带成分分析总结。蛋白浓度隔离使用不连续的PercolWestern blot法测定L -蔗糖梯度。无蛋白（ - ）目前，0≥0.2（+），0.2≥0.8 (++),或&gt; 0.8 (+++)倍大于蛋白质上清（皮质匀浆，离心），代表目前在N = 3。 <img alt="计划1" src="/files/ftp_upload/3196/3196scheme1.jpg" /> 计划1:一个SN准备使用一个不连续Percoll蔗糖密度梯度的原理图，解剖小鼠大脑皮质，匀浆，并在低速离心除去非均质组织，细胞，整个细胞器， 如原子核和膜。不连续梯度应当有明确定义的层。插图显示的图片，仅供都被染成了蓝色，只有在为了形象化层的层，例如。上清分层不连续Percoll蔗糖梯度上，在中等速度离心。从渐变的SN频段，然后检索，并准备使用。 <img alt="图1" src="/files/ftp_upload/3196/3196fig1.jpg" /> 图1。匀浆分馏上的不连续Percoll蔗糖梯度 。鼠标皮质匀浆，上升至6阶或分数上不连续Percoll蔗糖梯度分离。纯化的，积极的SNS（*）被发现在第五级。 <img alt="图2" src="/files/ftp_upload/3196/3196fig2.jpg" /> 图2。 SN的筹备工作给予了SNS的异构混合保留突触前和突触后成分 。SN的准备从C57BL / 6小鼠（P13到P21的年龄）准备了一份电子显微镜执行先前所描述的，表明SNS保留突触前和突触后的元素。 6个红色箭头指向性突触后密度。比例尺为1微米。 <img alt="图3" src="/files/ftp_upload/3196/3196fig3.jpg" /> 图3。的SN制剂的免疫印迹分析表明，突触和神经元的标记保持在纯化的SNS（第五级），但其他标志物的丰度显着降低。上清免疫印迹的（S，离心皮质匀浆）和不连续频段1-6 Percoll蔗糖梯度被探测的肌动蛋白（β-的结构，装载控制），GFAP（星形），GP73（高尔基），HSC70（细胞质和核），核纤层蛋白B1（核），Prohibitin（线粒体），PSD95（突触后密度），SNAP25（突触），突触（突触）和β3-微管蛋白（神经元特异性）。带可视化使用风暴860 Phosphorimager，N = 3的代表。 <img alt="图4" src="/files/ftp_upload/3196/3196fig4.jpg" /> 图4。 SNS是活动的，展览通过[35S] -蛋氨酸掺入从头蛋白质的合成。SNS预平衡至37 ° C为10分钟。 SNS是未经处理或预处理后15分钟与40微米茴香霉素或1μM胡桃在37 ° C，[35S]此外，气象，20μL快临标签混合S35简易标签，加上或减去谷氨酸37 ° C 30分钟。使用皮尔斯的SDS - PAGE样品制备试剂盒的样品制备，15％的聚丙烯酰胺凝胶上运行，干燥和定量使用一个分子动力学风暴860 phosphorimager，N = 3的代表，± SEM。 <img alt="图5" src="/files/ftp_upload/3196/3196fig5.jpg" /> 图5。从非连续Percoll蔗糖梯度带5条所载从头蛋白质合成的最高水平。乐队4，5和第6和上清液（离心皮质匀浆）预平衡至37 ° C下10分钟。然后，[35S] -蛋氨酸，快临标签混合S35简易标签，增加了，再加上或减去谷氨酸在37 ° C为30分钟。使用皮尔斯的SDS - PAGE样品制备试剂盒的样品制备，15％的聚丙烯酰胺凝胶上运行，干燥和定量使用一个分子动力学风暴860 phosphorimager，N = 3的代表，± SEM。 <img alt="图6" src="/files/ftp_upload/3196/3196fig6.jpg" /> 图6。从过滤法准备的SNS包含更多的碎膜比蔗糖不连续Percoll梯度法准备的SNS少整个SNS。 SNS准备通过一系列如前所述随着孔径的过滤器匀浆皮质 。3,14-15的SNS等分是在一个不连续Percoll蔗糖梯度离心和过滤法编制相比同等数额使用蔗糖不连续Percoll梯度法的材料。 <img alt="图7" src="/files/ftp_upload/3196/3196fig7.jpg" /> 图7。从过滤法编写的SNS包含少从头蛋白质合成活性比SNS准备来回米的不连续Percoll蔗糖梯度法。 SNS准备通过一系列降低孔径的过滤器，如前面所述 ，3,14-15匀浆皮质不连续Percoll蔗糖梯度方法。 SNS都准备工作，预平衡至37 ° C下10分钟。然后，[35S] -蛋氨酸，快临标签混合S35简易标签，增加了，再加上或减去谷氨酸在37 ° C为30分钟。使用皮尔斯的SDS - PAGE样品制备试剂盒的样品制备，15％的聚丙烯酰胺凝胶上运行，干燥和定量使用一个分子动力学风暴860 phosphorimager，N = 3的代表，± SEM。 <img alt="图8" src="/files/ftp_upload/3196/3196fig8.jpg" /> 图8。从年轻小鼠（P13）的SNS有更大的比年纪较大的老鼠（P85）翻译活动 。SNS从不同年龄的小鼠使用Percoll不连续蔗糖梯度法，在37 preequilibrated ° C 10分钟准备，处理或与谷氨酸治疗中存在[35S] -蛋氨酸（快递PRO标签混合S35简易标签）为30分钟，扣冻结，后清理与皮尔斯的SDS - PAGE样品制备试剂盒。 SNS的12％SDS聚丙烯酰胺凝胶上，然后运行，干和分子动力学风暴860 phosphorimager，N = 3，± SEM代表成像。 P14小鼠SNS至少3倍以上的P85小鼠积极。

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没有利益冲突的声明。

不连续Percoll蔗糖梯度准备此处所描述的是一个快速，可靠的方法来隔离活跃的SNS，它可以用于各种突触传递的实验。此渐变的方法，它是根据Dunkley等人，3,4开发的方法是一种细胞内的脑分馏过程分离前和突触后膜囊泡与另一个相关的衍生。没有进一步纯化这些SNS兼容多种分子生物学技术，包括免疫，免疫印迹，流式细胞仪，监测从头蛋白质的合成，通过[35S] - MET注册成立后，电子?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>微BCA蛋白检测试剂盒</td><td>刺穿</td><td> 23235 </td><td></td></tr><tr><td> 氯化钙 </td><td>费舍尔</td><td> C79 - 500 </td><td></td></tr><tr><td> CO 2气体</td><td>公司Airgas（UW - MDS） </td><td>光盘50 </td><td></td></tr><tr><td> EDTA的</td><td>零售物价指数</td><td> E57020 </td><td></td></tr><tr><td>乙醇</td><td>费舍尔</td><td> A407SK - 4 </td><td></td></tr><tr><td>盐酸</td><td>费舍尔</td><td> A142 - 212 </td><td></td></tr><tr><td> Percoll </td><td> GE医疗集团</td><td> 17-0891-01 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td>费舍尔</td><td> P285 - 500 </td><td></td></tr><tr><td> PierceSDS - PAGE样品制备试剂盒</td><td>刺穿</td><td> 89888 </td><td></td></tr><tr><td>氯化钠</td><td>零售物价指数</td><td> S23020 </td><td></td></tr><tr><td>碳酸氢钠3 </td><td>费舍尔</td><td> BP328 - 500 </td><td></td></tr><tr><td>娜2保护海港条例"4 </td><td>费舍尔</td><td> S381 - 500 </td><td></td></tr><tr><td>蔗糖</td><td>零售物价指数</td><td> S24060 </td><td></td></tr><tr><td> Tris碱</td><td>零售物价指数</td><td> T60040 </td><td></td></tr><tr><td>河豚毒素</td><td>西格玛</td><td> T5651 </td><td></td></tr><tr><td>快临标签混合S35简易标签</td><td> Perkin Elmer公司</td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th>设备</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>解剖工具</td><td></td><td></td><td></td></tr><tr><td> Dounce匀浆，7毫升（带有两个玻璃杵标记为"A"和"B"） </td><td>惠顿</td><td></td><td></td></tr><tr><td> P1000的吉尔森Pipetman </td><td>吉尔森</td><td> F123602 </td><td></td></tr><tr><td> Allegra的6KR离心机</td><td> Beckman Coulter公司</td><td> 366830 </td><td></td></tr><tr><td>生长激素3.8转子，水平转子</td><td> Beckman Coulter公司</td><td> 360581 </td><td></td></tr><tr><td>贝克曼J2 - 21型离心机</td><td>贝克曼</td><td></td><td></td></tr><tr><td>贝克曼管与帽</td><td>贝克曼</td><td> 355672 </td><td></td></tr><tr><td>白色围墙适配器</td><td>贝克曼</td><td> 342327 </td><td></td></tr><tr><td>蓝壁适配器</td><td>贝克曼</td><td></td><td></td></tr><tr><td> JA - 17转子，固定角转子</td><td>贝克曼</td><td> 369691 </td><td></td></tr></tbody></table>用于免疫印迹抗体表: <table border="1"><tbody><tr><th>抗体名称</th><th>公司</th><th>目录编号</th><th>宿主物种</th><th>稀释倍数</th></tr><tr><td> β-肌动蛋白</td><td>西格玛</td><td> A5441 </td><td>小鼠</td><td> 1:2000 </td></tr><tr><td> GFAP </td><td>圣克鲁斯</td><td> SC - 65343 </td><td>小鼠</td><td> 1:200 </td></tr><tr><td> GP73 </td><td>圣克鲁斯</td><td> SC - 134509 </td><td>兔</td><td> 1:200 </td></tr><tr><td> HSC70 </td><td>圣克鲁斯</td><td> SC - 7298 </td><td>小鼠</td><td> 1:200 </td></tr><tr><td> Laminβ </td><td>圣克鲁斯</td><td> SC - 6261 </td><td>山羊</td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td>圣克鲁斯</td><td> SC - 28259 </td><td>兔</td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore公司</td><td> MAB1596 </td><td>小鼠</td><td> 5微克/μL </td></tr><tr><td> SNAP25 </td><td> Abcam公司</td><td> ab5666 - 100 </td><td>兔</td><td> 1:2000 </td></tr><tr><td>突触</td><td> Millipore公司</td><td> MAB368 </td><td>小鼠</td><td> 1:500 </td></tr><tr><td> β- 3 -微管蛋白</td><td>圣克鲁斯</td><td> SC - 80016 </td><td>小鼠</td><td> 1:200 </td></tr></tbody></table>

preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient

Synaptoneurosomes（SNS）小鼠大脑皮质的同质化和分馏后获得的。他们再封好，水疱或离开时，皮层组织匀浆脱离的轴突终端的隔离终端。 SNS的保留前和突触后的特点，这使得它们在突触传递的研究非常有用。他们保留用于神经信号的分子机制，并能够吸收，储存和释放神经递质。 积极SNS的生产和隔离可能会出现问题的使用聚蔗糖，可细胞毒性，并要求延长离心由于密度高，过滤和离心的方法，它可以在低活动导致机械损伤的SNS等媒体。然而，使用不连续Percoll蔗糖密度梯度分离SNS提供了一种快速的方法来产生良好收益的翻译活跃的SNS。 Percoll蔗糖梯度法快速，温和的，因为它采用等渗条件下，有越来越短的离心旋转，并避免离心步骤，颗粒SNS和造成机械损伤。

Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

一个准备从小鼠大脑皮质翻译活跃，完好synaptoneurosomes（SNS）的方法来描述。该方法采用一个不连续Percoll蔗糖密度梯度，允许快速制备活跃的SNS。

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

preparation-synaptoneurosomes-from-mouse-cortex-using-discontinuous

Research

JoVE Journal

Neuroscience

31.4K 次观看.  University of Wisconsin. 一个准备从小鼠大脑皮质翻译活跃，完好synaptoneurosomes（SNS）的方法来描述。该方法采用一个不连续Percoll蔗糖密度梯度，允许快速制备活跃的SNS。

视频: Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

调查深皮层的功​​能和皮层下结构采用立体定向脑电图:由前扣带皮层的经验教训

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...