这项工作得到了新的启动资金来自叶史瓦大学阿尔伯特爱因斯坦医学院的研究者RVS。

1。示踪剂在体内提供使用无菌手术（第1.1-1.16） 整个过程中，我们建议使用标准的无菌手术技术。这包括使用无菌手套，隔离衣和口罩。工具应进行高压灭菌或使用前彻底清洁的水，然后乙醇。在手术过程中，特别是与动物之间，干净的纸巾碎片珠用热灭菌（无菌250＃18000-45，精细科学的工具）的工具和干燥消毒仪器关闭。此外，至关重要的是，您组织您的工作空间，你有动物准备（剃毛等），手术和恢复区的动物可以很容易地监控，温暖，并与需要的镇痛药提供指定地点。生存手术成功的关键是，以减少感染的风险，并保持一个积极的手术后护理的策略。请参阅表在这里您可以鳍D所有必要的设备和试剂，本议定书。 <ol><li> Avertin（2,2,2 - Tribromoethanol，Sigma公司，圣路易斯MO;猫＃T48402）新鲜配制麻醉成年小鼠通过腹腔注射（IP）0.2ml/10grams体重的剂量。如其他麻醉药异氟醚或氯胺酮/甲苯​​噻嗪也同样有效，但确保您的实验室已经在使用前所需的机构和联邦批准。如果您使用的幼仔（P0 - P10），您可以麻醉碎冰10-15分钟。确保每个小狗的皮肤不直接接触与冰（例如，放置在乳胶手套的手指），因为冷水会迅速诱发致命的低温。确认的动物表演与动物的后爪钳或手指脚趾捏在接受麻醉平原。如果有一脚蹬反射，等到动物是这种刺激的反应迟钝，作为一个更深层次的指标麻醉平原。 </li><li>一旦动物完全麻醉躺在它的背侧与它的头远离你和它的尾巴面向您面临的无菌纱布垫。电推剪剃的皮毛，为了给自己一个清晰的手术视野足够宽，能够获得中枢神经系统。两套酒精擦拭，然后70％的酒精/优碘擦拭剃光区域，然后再次用酒精擦拭干净。清洁手术区时，使用内到外的圆形图案。您可以选择您的手术视野清楚划分削减了一个小洞，进入无菌纱布垫，放置在你的光头和清洁手术部位开幕。虽然保持无菌手术领域可以是棘手的工作时的小动物，这样做将有助于降低感染的风险。 </li><li>使用钳提起皮肤，并在较低的胸上腰椎ř以上直接中线切口，用剪刀egion。较低的胸部上腰区可以识别运行手指沿椎骨觉得在脊柱驼背（图1）。 </li><li>切开筋膜和肤浅的脊髓肌肉。如果有必要，停止任何出血烧灼血管，加利福尼亚州福斯特城，精细科学工具;猫＃18000-00。在幼崽，应用，切口周围用棉签温和的压力是足够的止血。 </li><li>删除一个或两个的下胸段棘突执行 - 上腰脊髓背椎板切除术，切割后对脊柱，关节突及背侧的棘突之间的中途两侧椎板（图2） 。要小心，不要破坏你的剪刀脊髓和总是取出小碎骨，因为他们可以划破脊髓。请注意，在产后早期幼崽的脊柱没有完全僵化，这意味着，骨骼都非常软，可以很容易地削减。因此，要注意不要剪得深，因为您可能损伤脊髓。 </li><li>如果你不喜欢在成年小鼠进行背椎板切除术，我们发现，你可以切割软组织之间的椎体节段的脊髓暴露不切割任何骨一个小区域。虽然这两种方法都是有效的路线进入到脊髓，避免背椎板切除术，可确保脊髓仍然完好无损。 </li><li>您的使用千分尺注射器的示踪剂（悬液0.2ml; Gilmont仪器猫填补一把拉住的玻璃电极（高硼硅玻璃毛细管;猫＃300056，哈佛仪器，Holliston，硕士，双级玻璃微管拖轮从Narishige 001 - PC - 10型） ＃GS - 1100）或等效的交付设备。 </li><li>拧紧到位玻璃微电极上的显微和，电极0.1 - 0.5mm的中间插入下胸椎脊髓上腰区中期线和脊髓的外侧缘。动物可能会在注射位点的准确识别的立体定位仪和稳定的动物（小动物脑立体定位仪型号940 - A和电极的机械手模型从Kopf仪器960）举行。另外，如果您的麻醉制度，可定制，实现了稳定的平面上，没有因深呼吸显著运动轻轻地录音下来的动物来就足够了。 </li><li>压力注入到脊髓超过3-5分钟，约WGA的Alexa的488（麦胚凝集素的Alexa Fluor 488共轭;猫＃W11261，Invitrogen公司，卡尔斯巴德，加利福尼亚）的2％的溶液0.5μL，WGA - Alexa的555 （麦胚凝集素的Alexa Fluor 555共轭;猫＃W32464，Invitrogen公司，卡尔斯巴德，加利福尼亚），WGA - Alexa的350（麦胚凝集素的Alexa Fluor 350共轭;猫＃W11263，Invitrogen公司，卡尔斯巴德，加利福尼亚），WGA HRP（麦胚凝集素辣根过氧化物酶;西格玛，圣路易斯，密苏里州猫＃L7017）或10％0.5μLBDA（生物素葡聚糖胺;猫＃D1956，Invitrogen公司，卡尔斯巴德，CA）缓冲磷酸盐（PBS;西格玛，圣路易斯，密苏里州;猫＃P4417）。注射示踪剂，结果这些卷标签大量的纤维。较小的卷将需要针对较小的原子核（或单个核分区域），每个调查员应凭经验确定理想的音量提供注射。离子透入，应考虑为精确的注射准确的纳升交付。我们通常用0.5％的快速绿色（Sigma公司，圣路易斯，密苏里州，猫＃F7252）补充所有示踪解决方案，在手术过程中的可视化的注射部位。 </li><li>我们添加玉米油的加入1​​μl（Sigma公司，圣路易斯，密苏里州，猫＃C8267）每BDA的解决方案中加入10μl注射前坚持拉移液器的墙壁，以防止混合。如果你有麻烦弹出示踪吸管，慢慢收回的提示创建一个在组织中的小口袋，然后再次尝试注射。这是不寻常的微电极尖端堵塞。在大多数情况下，口袋里还有助于创建一个水库周围的神经元可以吸收的示踪。请记住，总是弹出示踪缓慢，以避免在你的针附近的大规模的组织损伤。 </li><li>轻轻关闭皮肤伤口剪辑和组织胶。或者，您可以使用缝合关闭伤口。 </li><li>注意与实践的外科手术，至少直到此时，可以在不到20分钟完成。 </li><li>放置在恢复室（Vetcare室模型9.16.0猫。哈佛仪器＃340508），以避免低温，加快恢复的速度加热垫（电热垫猫。＃341241）从哈佛仪器的动物。另外，您可以使用许多使加热室的模型之一。监测呼吸频率和检查该动物是试图移动的迹象。大多数动物通常手术后10-15分钟内响应。 </li><l我&gt;动物从麻醉中恢复后，需要为他们提供水和食物的自由采食和止痛药。这是你的动物保持密切关注您的手术后护理制度的一部分，最重要的。定期检查的疼痛，不适，感染的迹象，足够再次进食和饮水等，请与您机构的动物福利工作人员和兽医讨论您的需求，制定最佳的协议，成功地完成你的实验，并保持必要的照顾您的所有动物。 </li><li> WGA - 555 Alexa的WGA - Alexa的488，和WGA - HRP的快速运输在神经元中，我们发现，在双方产后早期和成年小鼠的Alexa结合示踪剂在24小时内强劲的标签长纤维束（Reeber等2011） 。 WGA - Alexa的350也迅速运虽然相比其他Alexa的染料，其知名度是高度依赖的过滤器设置的质量和灵敏度（数据未显示; Reeber等2011） </李&gt; <li>北京经济技术开发区，通常需要更长的生存时间，以充分跟踪神经预测，因此，脊髓小脑预测动物将需要大约11天之后牺牲的完整映射。 </li></ol> 2。长满青苔的纤维anterogradely追溯到检测<ol><li>在各自的生存期麻醉与Avertin（或您首选的麻醉剂）的小鼠。 </li><li>一旦有没有与0.1M PBS（PH7.2）灌流，血液通过心脏刷新的反射。然后，修复组织灌流4％多聚甲醛的PBS（PFA）的动物。此外你将分析的地区，它始终是必要的，以获得从注射部位注射是多么大的回顾性分析，显示电极造成的组织损伤的程度的组织，并确定在纤维可能标签通道（图3看到Mesulam。Reeber等，1982; 2011）。 </li><li> Postfix的的BR灌注小鼠在4％煤灰的24-48小时，然后ains cryoprotect蔗糖稀释在PBS（15％为1〜2小时，或直至组织下沉到容器的底部，然后30％过夜，或直至在缓冲解决方案组织汇）。组织，然后从蔗糖中删除，轻轻擦了擦用纸巾擦干，然后沉浸在组织华侨城模具（最佳切削温度;樱花精科美国公司，CA）。组织使用一个-80 ° C冰箱冷冻和储存在相同的温度，直到它准备好被削减。 </li><li> Alexa的荧光团后直接可见标签和不需要额外的染色。因此，灌注后的WGA Alexa的追查组织可以切割和成像上，或直接作为wholemount准备在4％PFA成像。我们发现组织在4％PFA给wholemounts追溯到预测的地形成像的最佳折射率成像。对于部分，切串行40微米厚的科伦人在低温恒温器部分和收集在PBS自由浮动部分。由于煤灰的毒性，确保一个适当的口罩戴在灌注和成像，保持通风良好的地方，并在不使用时立即返回任何PFA或PFA固定组织的密封容器。如果您的显微镜是在位于一个合适的通风柜的，它是理想。 </li><li> WGA - HRP标记的纤维，可以发现一个四甲基联苯胺（TMB）显色。我们不会描述HRP染色协议，因为它以前曾详细描述（沃格尔和Prittie 1994年。米利托等人2010） </li><li>对于BDA标记纤维孵育2小时在链霉亲和素- CY3自由浮动的组织切片（货号＃434315，Invitrogen公司，卡尔斯巴德，加利福尼亚）或链霉亲和的Alexa Fluor 488或555（货号为488和猫S11223 S21381为555，Invitrogen公司，卡尔斯巴德，加利福尼亚州），在PBS洗，然后装入氟凝胶。对于民建联，孵化组织切片ABC反应缓冲液过夜（货号＃PK - 4000，Vector实验室，公司伯林格姆，CA），在PBS漂洗3次，然后反应5-10分钟，在DAB（0.5mg/ml的矢量Laboratories公司，Burlingame的CA），辅以加入10μl30％H 2 O 2每50毫升DAB溶液。 </li></ol> 3。免疫组化<ol><li>免疫组化法进行如前所述（米利托等人，2008年）。简单地说，在0.1M PBS孵化组织切片中含有10％的农工商（NGS; Sigma公司，圣路易斯MO），0.1％Tween - 20的主要抗体在室温为16-18小时（见下文）。洗净组织切片在PBS的3倍，然后孵育二抗在室温下2小时最大（见下文）。再次冲洗组织和揭示了免疫反应，如下所述。 </li><li>为了确定我们共同标记的自由浮动，使用传统方法的双重染色的组织切片的Purkinje细胞和长满青苔的纤维之间的关系。我们使用WGA - Alexa的555跟踪和Alexa 488标记的驴抗鼠抗体（Invitrogen公司/分子探针公司，卡尔斯巴德，CA）检测ZebrinII（1:250;，卡尔加里，加拿大卡尔加里大学博士理查德霍克斯，实物礼品）。 ZebrinII是一种鼠单克隆抗体被用来直接从花杂交瘤细胞培养基。在1:1500稀释的荧光标记的二抗。组织切片染色后安装在带电玻片，然后用Tris缓冲液（电子显微镜科学，哈特菲尔德，PA）氟凝胶或Vectashield hardset coverslipped安装介质用DAPI（货号＃H - 1200，媒介实验室，伯林格姆，CA）三色荧光成像。 </li><li>我们测试处理在初级抗体的情况下组织切片，特异性抗体。在这样的对照实验，检测无信号表明，我们观察到的染色是不是由于非特异性信号从第Ë Alexa的共轭抗体（数据未显示）。 </li></ol> 4。显微镜和数据分析<ol><li>我们捕捉的组织部分使用徕卡DFC360 FX（荧光）和DFC490（DAB和TMB反应组织切片）摄像机安装在徕卡DM5500显微镜的显微照片。 </li><li>我们采集和分析的组织部分，使用徕卡应用套件和徕卡应用套件FX软件包的图像。 </li><li> wholemounts显微照片被捕获使用徕卡DFC3000 FX相机安装在徕卡MZ16足总杯运行徕卡应用套件FX软件的体视显微镜。 </li><li>我们的显微镜都配备了徕卡CY3（＃11600231）和FITC（＃11513880）过滤器，和一个额外的A4的DAPI /紫外线过滤器（型号＃11504162）DM5500。 </li><li>收购后确定使用徕卡软件优化/曝光过度的图像。我们所有的原始数据导入到Adobe Photoshop CS4和如有必要，瓦特发送正确的每个图像的清晰度，亮度，对比度或水平只整场。其他照片编辑软件，可以用来作为首选。 </li><li>这里介绍的原理图绘制在Adobe Illustrator CS4。 </li><li> Alexa的555和CY3染色的神经元，这是用我们的过滤器检测为红色，在整个手稿提出的图像伪彩色洋红。 </li></ol>动物所有的动物实验研究批准的IA​​CUC动物协议下进行，根据阿尔伯特爱因斯坦医学院的机构准则。男性和女性的远交瑞士韦伯斯特（泰康利，奥尔巴尼，纽约州）或自交系C57BL6J（JAX，酒吧港，ME）的小鼠均保持在我们的殖民地，用于所有的研究。阴道插件检测当天中午被认为是胚胎每天0.5。 5。代表性的成果： 脊髓小脑传入终止到parasagi在小脑ttal带（图3; Voogd等人，1969年; Grishkat和1995年艾森曼，1994年Vogel和Prittie; VIG等2005年; 2009年Apps和霍克斯;米利托等2010;。Reeber等2011）。较低的胸上腰椎脊髓，以证明我们注入WGA - Alexa的示踪剂：1）可以使用这些示踪剂一贯标签的特定子集的终端在小脑（图3; Reeber等2011），2）。 WGA - Alexa的示踪剂，是强烈的明亮，可用于可视化整个小脑苔藓纤维地形的总体格局（图3; Reeber等，2011），3），可以组合使用WGA - Alexa的488和555跟踪多个大片在同一动物（图4），4），WGA - Alexa的示踪与免疫组化染色（图5）兼容。在最近的工作中，我们发现，WGA - Alexa的示踪剂，在早6小时后提供的示踪到脊髓小脑标签终端和是一个很好的选择跟踪的architecturE发展的途径（Reeber等。2011年）。 <img alt="图1" src="/files/ftp_upload/3371/3371fig1.jpg" /> 图1提供一个我们的手术区的形象 。较低的胸上腰椎区域，可沿椎骨运行你的手指感到在脊柱驼背确定示踪剂在体内。答：设备安装。背侧椎板切除应大致在中间的“驼峰航线”，这是由黄色的箭头表示。 <img alt="图2" src="/files/ftp_upload/3371/3371fig2.jpg" /> 图2插图的一个较低的胸-上腰椎段背椎板切除前后 A.一个完整的从较低的胸上，木材鼠标脊柱椎体段的原理B.一个dorsa升椎板切除术是由一个或两个椎体节段的棘突，消除切割之间的关节突和背棘突椎板中途后执行。 <img alt="图3" src="/files/ftp_upload/3371/3371fig3.jpg" /> 图3。WGA - Alexa的示踪剂，是强烈的明亮，可用于可视化传入高分辨率投影地形。示意图说明WGA - Alexa的起源和终止555标记的脊髓小脑的神经元。B.后注射成人脊髓上到下胸腰椎地区提供555 WGA - Alexa的网站的形象 。C. Wholemount形象D. WGA - Alexa的555顺行追踪小脑束后，下胸椎脊髓上腰区的WGA Alexa的555注射前小叶。揭示长满青苔的纤维带，共同ronal组织部分。小叶被定义为罗马数字和中线（适用于所有图像）两边的数字标签苔藓纤维带。比例尺：B，500微米彗星ð 500微米，200微米。 <img alt="图4" src="/files/ftp_upload/3371/3371fig4.jpg" /> 图4。WGA - Alexa的示踪标记多个神经通路在同一动物的有效率 。 A. Wholemount示意图鼠标总结小脑脊髓小脑苔藓纤维带的总体格局。 ，无论是WGA的Alexa的示踪剂，WGA - Alexa的555右侧的腰椎脊髓和WGA Alexa的488注入到同一网段左侧 B.日冕组织切条通过后小叶运到小脑和成功标记的终端不同的子集（见也Reeber等2011）。缩写：摩尔cular层（ML）;浦肯野细胞层（PCL）;颗粒细胞层（GCL）;白质（WM）。比例尺：B，100微米。 <img alt="图5" src="/files/ftp_upload/3371/3371fig5.jpg" /> 图5。WGA - Alexa的示踪剂可用于神经元和预测的三重标记免疫组化和组织学相结合。 A. WGA - Alexa的555是存放在小叶三苔藓纤维终端B. ZebrinII染色显示小叶三数组的浦肯野细胞的条纹。C. DAPI荧光染色的神经元和胶质细胞的细胞核。D.板，A，B合并图像和彗星显示传入乐队，浦肯野细胞的条纹，和一般细胞构筑三重标签。EH。高倍率图像显示板公元盒装地区。浦肯野细胞的条纹编号如前所述（审查（2009年Apps和霍克斯））。缩写：分子层（毫升）;浦肯野细胞层（PCL）;颗粒细胞层（GCL）。比例尺：500微米（AC），H，500微米（EG）。

<ol>
	<li>Apps, R., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+cortical+organization:+a+one-map+hypothesis.">Cerebellar cortical organization: a one-map hypothesis.</a> Nat. Rev. Neurosci. 10, 670-681 (2009).</li><li>Grishkat, H. L., Eisenman, L. M. <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+spinocerebellar+projection+in+the+prenatal+mouse.">Development of the spinocerebellar projection in the prenatal mouse.</a> J. Comp. Neurol. 363, 93-108 (1995).</li><li>Mesulam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tracing neural connections with horseradish peroxidase. , (1982).</li><li>Reeber, S. L., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+expression+of+a+cocaine-+and+amphetamine+regulated+transcript+(CART)+peptide+reveals+complex+circuit+topography+in+the+rodent+cerebellar+cortex.">Patterned expression of a cocaine- and amphetamine regulated transcript (CART) peptide reveals complex circuit topography in the rodent cerebellar cortex.</a> Journal of Comparative Neurology. , (2011).</li><li>Reeber, S. L., Gebre, S. A., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+mapping+of+afferent+topography+in+three+dimensions.">Fluorescence mapping of afferent topography in three dimensions.</a> Brain. Struct. Funct. , (2011).</li><li>Sillitoe, R. V., Stephen, D., Lao, Z., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engrailed+homeobox+genes+determine+the+organization+of+Purkinje+cell+sagittal+stripe+gene+expression+in+the+adult+cerebellum.">Engrailed homeobox genes determine the organization of Purkinje cell sagittal stripe gene expression in the adult cerebellum.</a> J. Neurosci. 28, 12150-12162 (2008).</li><li>Sillitoe, R. V., Vogel, M. W., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engrailed+homeobox+genes+regulate+establishment+of+the+cerebellar+afferent+circuit+map.">Engrailed homeobox genes regulate establishment of the cerebellar afferent circuit map.</a> J. Neurosci. 30, 10015-10024 (2010).</li><li>Vig, J., Goldowitz, D., Steindler, D. A., Eisenman, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentation+of+the+reeler+cerebellum:+segregation+and+overlap+of+spinocerebellar+and+secondary+vestibulocerebellar+fibers+and+their+target+cells.">Compartmentation of the reeler cerebellum: segregation and overlap of spinocerebellar and secondary vestibulocerebellar fibers and their target cells.</a> Neuroscience. 130, 735-744 (2005).</li><li>Vogel, M. W., Prittie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographic+spinocerebellar+mossy+fiber+projections+are+maintained+in+the+lurcher+mutant.">Topographic spinocerebellar mossy fiber projections are maintained in the lurcher mutant.</a> J. Comp. Neurol. 343, 341-351 (1994).</li><li>Voogd, J., Broere, G., van Rossum, 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+medio-lateral+distribution+of+the+spinocerebellar+projection+in+the+anterior+lobe+and+the+simple+lobule+in+the+cat+and+a+comparison+with+some+other+afferent+fibre+systems.">The medio-lateral distribution of the spinocerebellar projection in the anterior lobe and the simple lobule in the cat and a comparison with some other afferent fibre systems.</a> Psychiatr. Neurol. Neurochir. 72, 137-151 (1969).</li><li>Wu, H. S., Sugihara, I., Shinoda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+patterns+of+single+mossy+fibers+originating+from+the+lateral+reticular+nucleus+in+the+rat+cerebellar+cortex+and+nuclei.">Projection patterns of single mossy fibers originating from the lateral reticular nucleus in the rat cerebellar cortex and nuclei.</a> J. Comp. Neurol. 411, 97-118 (1999).</li></ol>

没有利益冲突的声明。

我们已经描述了手术和技术的成功使用一种新型的荧光为基础的方法，迅速在发展中国家和成年小鼠的神经预测标签的轴突和树突跟踪所需的详细信息。使用WGA - Alexa的我们展示了如何示踪剂和标记可用于图案电路地形分析在高分辨率和在三个方面。 许多示踪剂追踪神经回路，包括霍乱毒素B亚基，生物胞素，Neurobiotin，菜豆leucoagglutinin（PHAL），fluororuby，fluoremerald和荧光金。?...

<table border="1"><tbody><tr><td> 设备/试剂 </td><td> 型号/目录编号 </td><td> 公司 </td></tr><tr><td>珠灭菌器</td><td>型号无菌250猫。 ＃18000-45 </td><td>精细科学工具</td></tr><tr><td> Cauterizer </td><td>猫。 ＃18000-00 </td><td>精细科学工具</td></tr><tr><td>硼硅玻璃毛细管</td><td>猫。 ＃300056 </td><td>哈佛仪器</td></tr><tr><td>双阶段玻璃微管拖轮</td><td>型号001 - PC - 10 </td><td> Narishige </td></tr><tr><td>千分尺注射器</td><td>猫。 ＃GS - 1100 </td><td> Gilmont仪器</td></tr><tr><td>小动物脑立体定位仪</td><td>型号940 - </td><td> Kopf仪器</td></tr><tr><td>电极的机械手</td><td>型号960 </td><td> Kopf仪器</td></tr><tr><td> Vetcare室</td><td>猫。 ＃340508 </td><td>哈佛仪器</td></tr><tr><td>加热垫</td><td>猫。 ＃341241 </td><td>哈佛仪器</td></tr><tr><td>徕卡DFC360 FX相机</td><td> DFC360外汇</td><td>徕卡</td></tr><tr><td>徕卡DFC490相机</td><td> DFC490 </td><td>徕卡</td></tr><tr><td>徕卡DM5500显微镜</td><td> DM5500 </td><td>徕卡</td></tr><tr><td>徕卡DFC3000 FX相机</td><td> DFC3000外汇</td><td>徕卡</td></tr><tr><td>徕卡MZ16 FA显微镜</td><td> MZ16足总杯</td><td>徕卡</td></tr><tr><td> CY3过滤器</td><td>型号＃11600231 </td><td>徕卡</td></tr><tr><td> FITC标记的筛选</td><td>型号＃11513880 </td><td>徕卡</td></tr><tr><td> A4的DAPI /紫外线过滤器</td><td>型号＃11504162 </td><td>徕卡</td></tr><tr><td>麦胚凝集素的Alexa Fluor 488共轭</td><td>猫。 ＃W11261 </td><td> Invitrogen公司</td></tr><tr><td>小麦GER米凝集素的Alexa Fluor 555共轭</td><td>猫。 ＃W32464 </td><td> Invitrogen公司</td></tr></tbody></table>

revealing neural circuit topography in multi-color

被组织成神经回路的功能地形图。为了形象直观地查看复杂的电路架构，我们开发了一个方法多种地形预测全球的图案，以可靠的标签。小脑是一个理想的的模型来研究神经回路的有序安排。例如，房室脊髓小脑长满青苔的纤维组织已经被证明是一个不可缺少的系统研究苔藓纤维图案的。我们最近表明，麦胚凝集素（WGA）的共轭Alexa的555和488可以用来跟踪，在发展中国家和成年小鼠的脊髓小脑苔藓纤维预测（Reeber等。，2011年）。我们发现了三个主要物业，使标签神经预测的WGA Alexa的示踪剂的理想工具。首先，Alexa的荧光团激烈，其亮度wholemount成像允许后直接跟踪。二，WGA - Alexa的示踪剂标签的整个发展中国家和成人神经projectio轨迹NS。第三，WGA - Alexa的示踪剂正在迅速运逆行及顺行方向。在这里，我们详细描述了如何准备示踪剂和其​​他所需的工具，如何进行手术治疗脊髓小脑跟踪和如何以最佳形象追溯到三个方面的预测。综上所述，我们提供了一个一步一步跟踪协议，将破译的组织和功能的地图不仅在小脑，而且在皮质，脑干和脊髓的连通。

Watch this Scientific Journal Video about Revealing Neural Circuit Topography in Multi-Color at JoVE.com

Revealing Neural Circuit Topography in Multi-Color

我们提供了一个实用指南提供示踪剂<em&gt;在体内</em&gt;，并使用作为一个模型系统的脊髓小脑通路证明成功在小鼠神经元电路分析的基本步骤。我们详细描述了我们全方位的跟踪协议，它利用麦胚凝集素（WGA）的共轭Alexa的荧光团。

多色揭示神经回路地形

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

revealing-neural-circuit-topography-in-multi-color

Research

JoVE Journal

Neuroscience

14.9K 次观看.  Yeshiva University. 我们提供了一个实用指南提供示踪剂在体内，并使用作为一个模型系统的脊髓小脑通路证明成功在小鼠神经元电路分析的基本步骤。我们详细描述了我们全方位的跟踪协议，它利用麦胚凝集素（WGA）的共轭Alexa的荧光团。

视频: Revealing Neural Circuit Topography in Multi-Color

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

睾酮和社会决策，结合行为内分泌和实验经济学：

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

为揭示复杂的大脑地形wholemount免疫组化

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

神经电路记录从一个完整的蟑螂神经系统

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

神经活动的传播的展开海马准备具有穿透微电极阵列

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...