我们要感谢他的科学意见和成像CIF显微镜设备UNIL平台特别是莫尼克Nenniger Tosato她出色的技术支持，以及让 - 伊夫Chatton。由瑞士国家科学基金会提供资助。

1。剖析鼠标VNO <ol><li>使用成年小鼠。由于信息素传感multimodalities牵连，小心株，基因型，性别和年龄之间的区别是重要的，会影响你的结果。在这里，我们选择成人VG的小鼠30以前用于识别信息素受体 ​​的一双（2 -庚酮，V1rb2）31（ 图1A） 。 </li><li>开始解剖之前，准备鲜冷人工脑脊髓液（学联; 118毫米氯化钠，碳酸氢钠3 25毫米，10毫米D -葡萄糖，氯化钾2毫米，氯化镁2毫米的NaH 2 PO 4 1.2毫米，氯化钙2 2毫米; pH值7.4）oxycarbon（95％O 2饱和：5％CO 2）。为此，除氯化钙混合所有学联组件。饱和与oxycarbon直接冒泡的解决方案。最后，添加氯化钙和DDH 2 0调整到所需的量。继续，直到ü冰学联解决方案SE。 </li><li>鼠标安乐死应优先做实验前颈椎脱位或CO 2吸入。 </li><li>剪下小鼠的头部。将其放置在寒冷学联解剖不断oxycarbonated显微镜下。 </li><li>切下颌，头部的位置，以可视化腭裂（ 图1B） 。 </li><li>在腭部的上半部分用微型剪刀（ 图1B）水平做一个切口，并删除与微观解剖钳腭膜。水合物和清洁暴露腔学联（ 图1C）。 </li><li>剪下的上部和下部的鼻中隔（ 图1C）。娇柔提取鼻中隔包含VNO和学联，它直接放置在冰（ 图1d） 。 </li><li>垂直分离VNO两部分组成，使用微解剖钳和微妙删除的犁鼻软骨胶囊（图1E）。确保所有的软骨片被删除。 </li></ol> 2。 VNO组织切片准备本节2）中所述的VNO组织切片准备，主要是钙成像调查。 VNO片也可用于浮动片immunohistostainings验证的组织结构的完整性（请参阅第3节）的协议）。 <ol><li>准备低熔点琼脂标准的PBS（3％）和您的工作场所。您将需要学联，切片和支持，微解剖钳，嵌入模具（22 × 22 × 20毫米），精密抹布，刷子，组织培养板，cyanacrylat胶，冰和温度计。 </li><li>填写3％低熔点琼脂嵌入模具。确保温度不超过41 ° C垂直放置在短期内消灭VNO（ 图2A），能够获得冠状切片。 </li><li>广场上的冰嵌入模具1分钟，直到凝固，然后将它单独从琼脂块。在金字塔形状的琼脂块切的地方用胶水切片机的支持（ 图2B）。 </li><li>切断与切片机冠状VNO片，0.5毫米/秒，0.7毫米的幅度和冷学联100微米厚度的速度和收集之前，他们在组织培养板填充用冷水的组织切片用毛笔学联。 </li><li>如果你的片包含像GFP的内源性荧光（在基因靶向神经元），你可以选择下一个荧光体视显微镜（图2C）。 </li></ol> 3。 VNO浮动片上的免疫组化此过程的目的是验证VNO组织切片获得第2）的完整性。乙酰-微管蛋白是微管/ VNO感觉神经元的树突和微绒毛细胞骨架的标记表示。钙离子成像实验，去方向TLY到第4节）的协议。浮动VNO片immunohistostaining方法可适应任何感兴趣的蛋白质表达的评价。为此，我们建议您1H修复VNO 4 ° C在固定液（4％多聚甲醛的PBS，pH值7.6）后的1.7点），在pH 7.6的协议，并从这个角度PBS使用，而不是学联的解决方案。 <ol><li>在组织培养板，固定片兴趣在固定液（4％多聚甲醛的PBS，pH值7.6）上半年在4 ° C。 </li><li>座在用含0.5％的农工商（正常山羊血清）10％和Triton X - 100解决方案的组织切片在4℃过夜。 </li><li>片与初级抗体（抗-乙酰-微管蛋白，鼠标，1:2000）孵育室温（RT）在PBS含0.25％的农工商5％和Triton X - 100的解决方案为16H。 </li><li>洗为5分钟，用PBS解决方案包含农工商2％的3倍。 </li><li>在黑暗中孵育与塞科ndary抗体（Cy5的标记的羊抗鼠，1:200）在用含农工商为1h，在室温下2％的解决方案。 </li><li> 5分钟，用PBS含有农工商2％洗3次，PBS农工商1％和PBS只。 </li><li>安装在一个荧光的安装介质（含有或不​​DAPI）片放置在疏水分隔区（使用疏水笔）的中心，并用盖玻片盖住片。 </li><li>用共聚焦显微镜和一个软件，让三维重建（图2D - F）的意见和收购。 </li></ol> 4。 VNO片钙成像<ol><li>下列程序将在黑暗的地方。孵育VNO片为1h，在37℃冷学联组成FURA - 2AM（7微米;股票1毫米的解决方案，在DMSO）装载解决方案C和聚醚0.1％（W / V; 2 DDH股票在20％以上的解决方案O）。 </li><li>保持在冰上直到使用oxycarbonation加载片。加载片可以保持3 - 4H。 </li><li>刷在灌注含有学联室装片和保持与组织切片锚（ 图3A）切片。 </li><li>广场上的钙成像显微镜（ 图3B）阶段的灌注腔，并在室温下连续灌注oxycarbonated学联。两极温度控制器的使用温度可适应。 </li><li>加载VNO片（ 图3C - E）的观测与倒置荧光显微镜，与25X或63X的客观和冷静的SNAP - HQ相机。照明是按顺序做（三百八十零分之三百四十零NM）与多色仪和记录在5HZ率。筛选的排放量在510 nm。细胞内钙的比例变化（ΔF= 340 nm/380纳米）的监测与合适的软件。 </li><li> Chemostimulation应选择根据你自己的实验目的。在这里，学联灌注含有信息素米IX（isobutylamine，2 -庚酮，4 -庚酮，2 -庚醇，pentylacetate，二甲基，α/β-金合欢烯，10 -6 M），2 -庚酮（10 -6 M）或新鲜采集的男性和女性（1 ： 1）鼠标尿（1:100）执行32。在实验结束时，灌注ATP（125微米）是作为一个可行性测试（图 3F），并应说明可行的神经元约80％的比例。在这些条件下，可以用来片长达2小时。 </li><li> GFP标记的感觉神经元亚群，可视具体照明（V1rb2表达神经元），可精确分析（ 图3G）。 </li></ol> 5。代表性的成果： 要建立一个功能检测，以调查多转导通路在小鼠犁鼻的地方，或确定新的信息素受体 ​​对，我们的VG鼠标线（图1A </stro吴&gt;）。在这条线，240 V1Rs受体之一，V1Rb2受体GFP标记允许特别的犁鼻感觉神经元亚群容易可视化。 VNO（ 图1E）的提取和清扫后，我们准备了急性VNO片（ 图2C） 。我们修复其中的一小部分，以检查和验证对乙酰化微管蛋白（图2D - F），由执行immunohistostainings tissular完整的准备。切片的其他一小部分用于钙离子成像实验。 ，切片装入FURA - 2AM（ 图3C - E），每个神经元细胞内钙离子水平作为ΔF比例（ 图3F）监测。 ATP（125微米），灌注后，迅速 ​​而短暂的ΔF的比例增加，预计（图3F）的神经元的可行性进行评估。尿液中的信息素的主要来源，灌注新鲜LY鼠标收集尿液（1:100）作为一种内源性的控制。它触发记录的神经元（细胞1和2， 图3F）在大约50％的钙瞬变。 GFP的照明下，V1Rb2阳性神经元观察到，灌注其称为信息素配体，2 -庚酮，启动细胞活化（细胞1，图3I）。有趣的是，神经元的激活也观察到在一小部分神经元不表达V1Rb2受体（GFP负神经元 ）（ 细胞 2，图3I），显示出复杂的犁鼻器信息素编码。 <img alt="图1" src="/files/ftp_upload/3311/3311fig1.jpg" /> 图1小鼠犁鼻器官的解剖。 （A）鼠标从VG线。 （b）在安乐死的鼠标，完整的头被放置在双目显微镜下，下颌被删除，以形象化的腭。小横切口完成（黑色虚线）。 （c）在拆除腭，鼻中隔内衬的VNO是削减在上部和下部（白色虚线），以方便VNO提取。 （四）VNO是定位在学联的解决方案，并分居（插入：更高的权力观两部分）。 （五）的犁鼻软骨胶囊微妙删除，并VNO无软骨胶囊，其余的实验中使用。比例尺：1毫米。 <img alt="图2" src="/files/ftp_upload/3311/3311fig2.jpg" /> 图2小鼠犁鼻急性片制备和tissular完整性。 （一）鼠标VNO是垂直嵌入在琼脂3％溶液。琼脂的温​​度必须低于41 ° C。 （二）琼脂块是金字塔形状和切100微米VNO部分（黑色虚线）在学联产生。 （三）VNO片，可以选择一个荧光体视显微镜下才能看到一个特定的标签的感觉神经元的人口。 （DF）Tissular完整，可以评估对乙酰化微管蛋白的蛋白质，一个更高的权力观V1rb2基因靶向神经元表现出完美的准备保护immunohistostainings。可以观察到其长期的树突表面到达管腔（五），以及在树突状旋钮和微绒毛的结构蛋白的表达神经元（F）比例尺是：B，5毫米，60微米，D，40微米，EF，20微米。 <img alt="图3" src="/files/ftp_upload/3311/3311fig3.jpg" /> 图3。钙成像和信息素在犁鼻急性组织切片检测。 （a）在一个FURA - 2AM装载阶段，VNO片放置在灌注室，并与维持一个适应的锚（高功率视图）。 （二）实验是在黑暗和VNO片进行连续灌注在室温真空系统与学联。温度可chosEN使用一个温度控制器系统组成一个双极佩尔蒂埃元素和热探头。这种特殊的实验是在室温下进行。 （CE）的VNO片霍夫曼相衬（HV）或前FURA 380纳米照明下观察（D）和（E与ATP）在神经元激活。 （六）神经元的生存能力评估灌注ATP（125微米）。在这里，chemostimulation尿（尿，1:100），或在165毫升/小时的流量恒定的鼠标信息素混合（混合pH值，10 -6 M）细胞内钙水平（ΔF）可以记录每个细胞（细胞1至3）。 （G）V1Rb2神经元是根据绿色荧光照明（1）可视化。 （H）加载这个神经元（1）以及非V1rb2表达神经元（2）所示。 （一）V1rb2神经元是能够与细胞内钙增加2 - 庚酮。这种特殊的非V1rb2表达神经元也响应2 - 庚酮（单元2）。比例尺：行政长官，20微米;生长激素，15微米;ΔF= 340纳米/ 380纳米。 （F和我）刺激应用程序的时间是表示，根据每个跟踪酒吧。任意颜色规模的荧光强度。

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P., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+evolution+of+the+V2r+vomeronasal+receptor+gene+repertoire+in+mice+and+rats.">Composition and evolution of the V2r vomeronasal receptor gene repertoire in mice and rats.</a> Genomics. 86, 306-315 (2005).</li><li>Levai, O., Feistel, T., Breer, H., Strotmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+in+the+vomeronasal+organ+express+odorant+receptors+but+project+to+the+accessory+olfactory+bulb.">Cells in the vomeronasal organ express odorant receptors but project to the accessory olfactory bulb.</a> J. Comp. Neurol. 498, 476-490 (2006).</li><li>Riviere, S., Challet, L., Fluegge, D., Spehr, M., Rodriguez, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formyl+peptide+receptor-like+proteins+are+a+novel+family+of+vomeronasal+chemosensors.">Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors.</a> Nature. 459, 574-577 (2009).</li><li>Liberles, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formyl+peptide+receptors+are+candidate+chemosensory+receptors+in+the+vomeronasal+organ.">Formyl peptide receptors are candidate chemosensory receptors in the vomeronasal organ.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 9842-9847 (2009).</li><li>Liman, E. R., Corey, D. P., Dulac, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRP2:+A+candidate+transduction+channel+for+mammalian+pheromone+sensory+signaling.">TRP2: A candidate transduction channel for mammalian pheromone sensory signaling.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 5791-5796 (1999).</li><li>Kelliher, K. R., Spehr, M., Li, X. H., Zufall, F., Leinders-Zufall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromonal+recognition+memory+induced+by+TRPC2-independent+vomeronasal+sensing.">Pheromonal recognition memory induced by TRPC2-independent vomeronasal sensing.</a> Eur. J. Neurosci. 23, 3385-3390 (2006).</li><li>Rodriguez, I., Feinstein, P., Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+patterns+of+axonal+projections+of+sensory+neurons+in+the+mouse+vomeronasal+system.">Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system.</a> Cell. 97, 199-208 (1999).</li><li>Boschat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromone+detection+mediated+by+a+V1r+vomeronasal+receptor.">Pheromone detection mediated by a V1r vomeronasal receptor.</a> Nature. Neuroscience. 5, 1261-1262 (2002).</li><li>Brechbühl, J., Klaey, M., Broillet, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grueneberg+ganglion+cells+mediate+alarm+pheromone+detection+in+mice.">Grueneberg ganglion cells mediate alarm pheromone detection in mice.</a> Science. 321, 1092-1095 (2008).</li></ol>

没有利益冲突的声明。

这里介绍的钙成像方法，使记录，在急性切片制备，小鼠犁鼻器神经元的信息素反应。通过这种方法，从动物解剖的犁鼻器神经元，一些做法，很容易实现，并提供了一​​个长期生活的组织准备。它允许像药理调查或研究的信息素编码耗时的实验。这种生理的技术，庞大的人口数量以及精确的神经元亚群可以具体分析。此外，这种方法可以很容易地适应其他钙染料为基础的免疫组织化学方法或实验。使用相结合的方法，必将使许多孤儿在小鼠犁鼻器官中表达的化学感受器的新的信息素配体的识别。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>腺苷三磷酸二钠溶液（ATP） </td><td> Sigma - Aldrich公司</td><td> A6559 - 25UMO </td><td></td></tr><tr><td>洋菜</td><td> Sigma - Aldrich公司</td><td> A7002 - 100G </td><td>优先免疫组化</td></tr><tr><td>琼脂（低熔点） </td><td> Sigma - Aldrich公司</td><td> A0701 - 25G </td><td>优先钙成像</td></tr><tr><td> CL - 100双极型温度控制器</td><td>华纳仪器</td><td> W64 - 0352 </td><td></td></tr><tr><td>共聚焦显微镜</td><td>徕卡显微系统</td><td> SP5的AOBS </td><td></td></tr><tr><td> Cy5的，AffiniPure山羊抗鼠IgG </td><td>杰克逊ImmunoResearch </td><td> 115-175-071 </td><td>防止光线照射</td></tr><tr><td> DMSO（二甲基亚砜） </td><td>默克公司</td><td> 317275 - 100ML </td><td></td></tr><tr><td>嵌入模具，剥离- A路</td><td> Polysciences </td><td> 18646A - 1 </td><td> 22 × 22 × 20毫米</td></tr><tr><td>荧光安装媒体（10毫升Vectashield用DAPI） </td><td> Rectolab </td><td> H - 1200 </td><td></td></tr><tr><td> FURA - 2AM </td><td> Teflabs </td><td> 0103 </td><td>防止光线照射</td></tr><tr><td> Glisseal N（真空润滑脂，60克） </td><td>三化螟化学</td><td> Glisseal ñ </td><td></td></tr><tr><td>大浴池录音室（RC - 26G） </td><td>华纳仪器</td><td> 64-0235 </td><td></td></tr><tr><td> MetaFluor（钙成像软件; v.7.6.5.0） </td><td> Visitron系统</td><td></td><td></td></tr><tr><td>单克隆抗微管蛋白，乙酰ð小鼠抗体</td><td> Sigma - Aldrich公司</td><td> T6793 - .2ML </td><td></td></tr><tr><td>正常山羊血清（NGS，10毫升） </td><td> Interchim </td><td> UP379030 </td><td></td></tr><tr><td>商会的平台（P - 1型） </td><td>华纳仪器</td><td> 64-0277 </td><td></td></tr><tr><td>聚醚F - 127，2克</td><td> Invitrogen公司</td><td> P - 6867 </td><td></td></tr><tr><td> ROTI科尔（计量瓶，10克） </td><td>卡尔罗斯</td><td> 0258.1 </td><td></td></tr><tr><td> SC - 20型双列直插式溶液加热器/冷却器</td><td>华纳仪器</td><td> W64 - 0353 </td><td></td></tr><tr><td>切片锚（SHD - 26GKIT） </td><td>华纳仪器</td><td> 64-0266 </td><td></td></tr><tr><td> Stereofluorescence显微镜</td><td>徕卡显微系统</td><td> MZ16FA </td><td></td></tr><tr><td>超级PAP PEN（疏水笔） </td><td>派尔国际</td><td> 22309 </td><td></td></tr><tr><td> TRITON X - 100 </td><td> Fluka公司</td><td> 93420 - 250ML </td><td></td></tr><tr><td>振动刀片切片机的VT 1200S </td><td>徕卡显微系统</td><td> 14048142066 </td><td></td></tr><tr><td> VisiChrome高速多色系统</td><td> Visitron系统</td><td></td><td></td></tr><tr><td>三维数据可视化（Imaris; v.6.3.1） </td><td>位平面科学软件</td><td></td><td></td></tr></tbody></table>

imaging pheromone sensing in a mouse vomeronasal acute tissue slice preparation

彼得Karlson和马丁Lüscher的使用，在1959年1的长期性信息素，首次描述用于种内通讯的化学品。信息素是易失性或非易失性的短命分子2分泌和/或在生物体液，如尿液，已知是信息素的主要来源的液体3,4中。在一个关键的动物模式，如亲属关系5,6， 分层组织 3和7,8性互动的各种信息素通讯牵连，因此直接与特定物种的生存9,10,11相关。在小鼠实验中，能够检测信息素主要介导犁鼻器（VNO）10,12，位于鼻腔的基础配对结构，并在软骨胶囊内。每个VNO有一个管腔13,14允许与外界接触的管状化学世界。感觉神经上皮主要由犁鼻器双极性感觉神经元（VSNS）15。每个VSN的一个单一的枝蔓在一个大的树突状旋钮轴承化学检测16牵连的100微绒毛向管腔结束。 VSNS许多亚群存在。他们表达，从而可能他们认识到17,18的配体（S）的化学感受器，它们是有区别的。两大犁鼻器受体的家庭，V1Rs和V2Rs 19,20,21,22，240 23 120 24名成员组成，分别在单独的层的神经上皮表达。嗅觉受体（ORS）25和甲酰肽受体（FPRs）26,27也表示VSNS。 无论这些神经元亚群，利用遥感信息素相同的下游信号通路是未知的。尽管起到透水钙通道（主要作用TRPC2）在28 TRPC2独立的传导渠道，成熟的神经元的微绒毛已建议 6,29 。由于神经元亚群的数量和特有的器官形态，在VNO目前的信号元素的药理和生理调查是复杂的。 在这里，我们目前的鼠标VNO急性组织切片进行钙成像调查的准备。这种生理方法允许意见，在活组织的天然环境，以前加载FURA - 2AM，钙染料的VSNS一般或个别亚群。这种方法也方便GFP标记的信息素受体的研究，是适应使用其他荧光钙探针。作为一个例子，我们在这里使用一个VG鼠标30行，在这种信息素V1rb2受体的翻译与一个多顺反子策略，以GFP表达。 </p&gt;

Watch this Scientific Journal Video about Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation at JoVE.com

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

在小鼠体内，能够检测信息素主要介导犁鼻器（VNO）。在这里，描述一种急性组织VNO切片进行钙成像的准备。这种生理方法允许生活在一个组织中的亚群和/或单个神经元的观察和受体 - 配体识别的方便。

成像遥感信息素在小鼠犁鼻急性组织切片的制备

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

imaging-pheromone-sensing-mouse-vomeronasal-acute-tissue-slice

Research

JoVE Journal

Neuroscience

16.1K 次观看.  University of Lausanne. 在小鼠体内，能够检测信息素主要介导犁鼻器（VNO）。在这里，描述一种急性组织VNO切片进行钙成像的准备。这种生理方法允许生活在一个组织中的亚群和/或单个神经元的观察和受体 - 配体识别的方便。

视频: Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

多电极阵列的犁鼻上皮录音

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

科研

神经科学

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 ...

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 ...

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

切片的制备，组织器官型培养，并记录在视交叉上核时钟基因活性的荧光素酶

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

在犁鼻器官的切片准备工作表示一个基因编码的钙传感器成像神经元的反应

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

切片热门：成人急性脑切片在生理温度

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...