这项工作是支持由美国国立卫生研究院（DC007681 AB），CT干细胞（AB）资助，:帕蒂基金会（AB），博士前露丝属Kirschstein国家研究服务奖（NRSA），（SZY），和NSF研究所的研究补助奖学金（BL）。 ，我们感谢Bordey实验室的手稿上了宝贵的意见。本发明的材料是基于部分由康涅狄格州康涅狄格州干细胞研究资助计划下的工作。它的内容是完全的责任作者，并不一定代表官方意见的康涅狄格州，康涅狄格州或CT创新国家公共卫生部，注册成立。

1。制备的解决方案，夹层，Vibratome的<ol><li>解，必须在正确的渗透压和pH值（ 表1）制备。人工脑脊液（ACSF）相比，剥离溶液与较低浓度的钠和钙，和更高浓度的镁制备。这是为了最大限度地减少在切割时的兴奋性毒性的影响。 </li><li>是饱和的夹层和学联解决方案都必须用95％O 2/5％CO 2鼓泡至少10分钟，以达到所需的pH值7.3。 </li><li>准备一盘冰浴中。在冰浴中放置一个夹层板，并填写解剖的解决方案。泡沫夹层板。 </li><li>准备的vibratome通过创建在冰浴中。将切片菜，在冰浴，填补了菜与，解剖的解决方案，和泡沫。 </li><li>填充片室学联和气泡，以确保该解决方案充分曝气，无论片被放置的位置。 </li></ol> 2。去除脑组织急性的大脑切片往往是健康与年轻小鼠。在啮齿类动物中产后SVZ的细胞结构是成熟的周围日龄（P）20 1。因此，我们试图限制我们的实验小鼠岁的P20-P30，但我们已经进行了成功的实验小鼠旧如旧3个月。在整个处理的组织，必须注意尽量减少机械运动和压力的大脑，保持冷却的条件下，暴露在SVZ尽快解决以下牺牲。 <ol><li>注射戊巴比妥钠后，鼠标迅速断头。被删除的皮毛，并切口是通过颅底。的头部，然后放置在填充夹层溶液的托盘。 </li><li>在稳定钳头，连续切削的周围的头骨，有时可达中线力显微镜issors。要小心，尽量减少接触的组织。在嗅球是从SVZ的新生神经元的最终目的地，必须注意维护该地区，尤其是在这个地区周围的骨切除。 Microscissor削减周围的颅骨背侧，允许容易拆除的头骨。 </li><li>现在，除去覆盖在大脑颅骨分离从下方大脑的骨头。如果在上一步中作出正确的削减，可以轻易删除此覆骨钳或镊子。 </li><li>虽然仍保持头用钳子，一个可以使用的外科刀片干净地除去几件之前的脑组织切片，如以下。一个冠状切口可以在水平的lambda。矢状削减可以作出的大脑的SVZ外侧的两侧上。一个保守的估计，从中线〜2.5毫米。如果需要的话，还可以被切成两半大脑这里。这些削减都会使几itate安装该组织，并允许更快地达到的SVZ脑切片时。重要的是要记住，施加少量的机械力对大脑的（ 如不推或拉）。 </li></ol> 3。急性脑片的制备<ol><li>为了准备随后的步骤，确保用于安装的超级胶水组织是免费的木屐和准备申请的vibratome板。重要的是要尽可能快而不损坏大脑。 </li><li>从下面，从底层骨组织分离，同时分离神经的脑组织，然后可以挖出。 </li><li>该组织被安装和胶合板并和对上vibratome下的。由于喙尾解剖的SVZ，我们一直专注于矢状切片，因为它保留的神经母细胞的迁移途径，胶质鞘，血管，主要是在这个方向对齐。可以个月UNT的组织在中线或创建的使矢状削减的SVZ外侧的平坦表面上。一个可选的步骤，是将3％的琼脂糖块后面的组织，作为叶片的抵接片脱落。我们优先使用氰基丙烯酸酯为基础的超级胶水。 </li><li>刀片，用乙醇冲洗，以除去油，并用蒸馏水冲洗。将刀片上的刀片保持器。的刀座的vibratome的安装。 </li><li>调整vibratome设置切割组织，每片的厚度为250-350微米。重要的是要使用高频振动和低速切割。 </li><li>逐步穿过组织节。在嗅球的外观是一个很好的指标，一个是附近的SVZ最外侧的切片将不含有地区在矢状切片。寻找其他地标包括之下的SVZ位于胼胝体，脑室和浓缩。 SVZ将首先出现在多个横向sagitTAL片。 RMS将显示在内侧矢状切片。 </li><li>删除片含有SVZ用塑料巴斯德吸管尖端切断。转移的切片保持腔。我们经常会得到更多的片比，我们可以在一天的工作。然而，切片会有所不同的量的细胞中含有的SVZ。它有时是必要寻找通过几个切片找到一个想要的用于成像。 </li></ol> 4。的显微镜和染料的制备片需要一个小时的孵育时间学联恢复的解剖和切片。在这片恢复期间，可以进行几个步骤。 <ol><li>移液管的制备钙染料和/或药物的应用<ol class="lalpha"><li>玻璃移液管需要有2-3微米直径的前端的长度（约2毫米）和角度（〜16°，当吸液管保持器），以防止与灌注室接触的前端，并允许用于放置的房间的目标。 </li><li>通常，人们可以制备6-8压力移液器的每一天的实验。 </li></ol></li><li>钙染料的制备<ol class="lalpha"><li>当制备的工作溶液（4-5μM由浴，250μM的压力的应用程序），它最好是暴露的最小量的DMSO的细胞。可以实现这一点，通过使高度集中的股票。例如，对于型Fluo-4，一管含有50μg每天使用。一个可以让本等分试样的10mM库存通过加入4.6微升的Pluronic F-127的20％的DMSO溶液。 </li></ol></li><li>准备显微镜设置（ 图2）。 <ol class="lalpha"><li>使用蠕动泵解决方案流有助于降低图像漂移。运行蠕动泵，以允许前学联通过灌注线运行，并确保它是无气泡。 </li><li>设置管灌注室入口，并确保没有泄漏。 </li><li>真空尖端的位置为ens尤尔溶液被吸入从灌注室。确保它是在一个适当的水平，使该计划的目的是沉浸在适当的工作距离，但没有那么高，这样的解决方案可能会蔓延室。低水平的解决方案也将减少流量的扭曲。一个恒定的愿望，也可以验证通过监听稳定性。 </li></ol></li></ol> 5。染料吸附量此方法已被详细描述，在另一原稿2。请见图其中。 <ol><li>巴斯样染料<ol class="lalpha"><li>做成1-2毫升的染料的工作溶液。对于氟上午04时，浓度为4-5μM是足够的标签SVZ神经母细胞。 </li><li>先转移到一个35毫米培养皿中的一个网底，已经充满学联片。更换由使用3毫升塑料移液管轻轻取出学联的解决方案，同时增加的calcium染料的解决方案。 </li><li>在37℃下孵育30-60分钟，在5％CO 2气体的受控环境。 </li><li>将切片成为学联填充的控股室，还没有进入细胞的染料洗去。或者，可以把它直接在显微镜灌注室运行学联。清洗周期是30-45分钟。 </li><li>切片转移到灌注室。 </li></ol></li><li>压力的染料中的应用<ol class="lalpha"><li>从室内转移切片，在显微镜下设置的灌注室。 </li><li>填充玻璃吸管与工作解决方案，为250微米，地方上的显微操作。 </li><li>放下移液器感兴趣的区域。可以放置吸移管的前端可以是几微米以上的片表面或埋几微米以下的片表面，但远离记录区。的移液管的角度没有AFFE克拉的装载效率。当拍摄的片表面之下，我们发现，将吸头的成像平面以下我们所期望的更好的成像面积将标签。 </li><li>当压力施加染料，的Picospritzer设置，以便它是&lt;3 psi的。我们没有预计染料扩散的体积的，因为这是受变异性吸液管尖端直径，施加压力和密度的组织处的染料注入。我们采用简单的，直到我们达到我们想要的负载评估的眼睛。切片损坏最小化，在应用过程中，尤其是当切片表面的前端的下方放置。申请1-2分钟。根据标签的眼睛评估，有时重复的应用程序是必要的。对于这个浓度和应用的持续时间，我们发现，表面的染料中的应用优先标记的神经母细胞，而在地表以下应用程序优先标记星形胶质细胞。然而，&gt; 3分钟在500μM中的应用也将标记成神经细胞无论是否尖端的上方或下方的片表面（JC Platel，未发表的观察）。允许恢复为30-45分钟洗净，切片。 </li></ol></li></ol> 6。共焦成像的活性钙<ol><li>查找感兴趣的区域先用一个低功耗的诸如4倍或10倍的目标。放置在中心的字段，然后再切换到更高的功率目标。在这一点上，人们可以评估一般切片微分干涉相差（DIC）下注意到SVZ细胞的外观的健康。健康的细胞呈圆形和丰满。此外，健康的细胞显示微弱的荧光-4，应该全部或明亮的荧光（死细胞）在无负载的负载。 </li><li>降低水浸入到感兴趣的区域上的目标，这是现在加载染料。 40倍或60倍的目标已经足够满足我们的需要，，虽然40X提供了一个更宽广的视野，并允许更大的吸管访问。当成像低于表面上，我们经常去片表面的3维结构和细胞的健康更好地保留至少15微米以下。 </li><li>验证灌注和真空胜任工作。 </li><li>设置在显微镜，这样的时间推移的分辨率和空间分辨率被设置为所需的速率。共聚焦显微镜，这些因素必须被平衡，作为时间分辨率的增加通常导致空间分辨率的损失，由于系统的扫描性质。活性钙，我们拍摄512x512像素的分辨率大约每秒一帧。如果单独收购的多渠道是必要的，这也将影响采集速率。设置电影的持续时间（2-10分钟）。 </li><li>启动运行。如果要进行药理研究，洗的拮抗剂或非脱敏激动剂的的至少5-10分钟，然后再作5-10分钟的电影。可能脱敏受体的激动剂可以敏锐地施加，例如通过使用某essure显微操作所控制的移液管。 </li></ol> 7。钙分析分析如下标准程序，我们描述了在透水出版物2-4。 F 0（ 即基线）和F是测得的平均荧光强度分别在整个感兴趣区中的所有的区域，并在每个ROI，。荧光的变化在被认为是如果它是&gt; 15％，F / F 0增加的Ca 2 +增加。计算细胞内Ca 2 +的变化使用Calsignal 5。 8。代表性的成果我们已经能够获得选择性加载SVZ细胞根据上述和其他地方2-4,6染料加载协议。浴加载中Fluo-4 AM（4-5或250μM），标签主要是神经母细胞在切片表面施加压力。虽然压力应用程序可以加载neurob持续更迅速地比浴加载在一个单一的片，清洗负载可以同时加载多个切片。 （1）确认身份的标记细胞神经母细胞是否显示4,7迁徙行为，（2）它们的形态，SULFORHODAMINE 101（3）染色阴性，星形胶质细胞的特定的染料3或（4）阳性DCX-红色荧光蛋白表达（JC Platel，未发表的观察）。成神经细胞迅速转移（平均为60微米/小时）在生理温度下4,7-9中 ，但它们的移动，可以容易地观察到，即使在室温下。运动神经母细胞并不构成一个问题，关于将地区的利益（投资回报率），因为我们的电影通常持续时间短，跟踪活性钙。然而，在漫长的等待期，如在药液的交流中，研究者必须小心，以配合在控制和治疗期间的投资回报率。这是不寻常的神经母细胞迁移进入或离开的我<html> maging场或焦平面。 压力中Fluo-4 AM（250μM）深成片和有限的时间（2分钟）优先标记SVZ星形胶质细胞。星形胶质细胞标记已被证实与SULFORHODAMINE 101的正面标签及其形态，尤其是存在对血管的预测和endfeet的的我们最近描述3。使用这些方法，我们观察到自发活动的SVZ神经细胞和星形胶质细胞的人口（ 图1）。星形胶质细胞的活动往往需要从事血管3波的形式。此外，使用钙成像的分析，药理激动剂的应用已发现或确认表达的GABA 受体对成神经细胞和神经胶质细胞6，AMPA受体和NMDA受体在神经母细胞4。 图"SRC ="/ files/ftp_upload/4071/4071fig1.jpg"/&gt; 图1。在星形胶质细胞活性钙传播。（A）代表平均时间的推移图像记录活性钙。星形胶质细胞在SVZ加载中Fluo-4的应用与压力是深成片的。电影被收购在0.75的时候步骤。感兴趣区（ROI）被放置在细胞活动。 （B）从感兴趣区的轨迹放置在细胞从（A）中所示的电影。过滤的移动平均线和标准化的痕迹。的垂直刻度表示2×ΔF/ F 0其中F是信号强度，和F 0是平均基线信号和ΔF= FF 0。 <img alt="图2" src="/files/ftp_upload/4071/4071fig2.jpg" /> 图2。照片灌注系统的管的一端被浸没在95％O 2/5％CO 2气体对erfused的解决方案。由蠕动泵（未示出），然后将溶液通过管和浴室灌注。的另一端被插入到安装在一个平台上的浴室的溶液入口。的诉求尖端，然后连接到该室的溶液出口和设置在一定的水平，以确定溶液高度。从电源插座中，抽吸尖端连接到真空管线，该抽吸从腔室到废物容器中的溶液。的灌注系统显示，视觉清晰度的显微镜载物台。

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M., Henschel, O., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP-expressing+cells+in+the+postnatal+subventricular+zone+display+a+unique+glial+phenotype+intermediate+between+radial+glia+and+astrocytes.">GFAP-expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes.</a> Glia. 54, 394-410 (2006).</li><li>Wang, D. D., Krueger, D. D., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+and+ionic+signature+of+neuronal+progenitors+of+the+postnatal+subventricular+zone+in+situ.">Biophysical properties and ionic signature of neuronal progenitors of the postnatal subventricular zone in situ.</a> J. Neurophysiol. 90, 2291-2302 (2003).</li><li>Tian, L., Hires, S. A., Mao, T., Huber, D., Chiappe, M. E., Chalasani, S. H., Petreanu, L., Akerboom, J., McKinney, S. A., Schreiter, E. R., Bargmann, C. I., Jayaraman, V., Svoboda, K., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+activity+in+worms,+flies+and+mice+with+improved+GCaMP+calcium+indicators.">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nat. Methods. 6, 875-881 (2009).</li><li>Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expanded+palette+of+genetically+encoded+Ca(2)+indicators.">An expanded palette of genetically encoded Ca(2) indicators.</a> Science. 333, 1888-1891 (2011).</li><li>Shigetomi, E., Kracun, S., Sofroniew, M. V., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+targeted+optical+sensor+to+monitor+calcium+signals+in+astrocyte+processes.">A genetically targeted optical sensor to monitor calcium signals in astrocyte processes.</a> Nat. Neurosci. 13, 759-766 (2010).</li></ol>

没有利益冲突的声明。

SVZ细胞钙成像已被用于研究在神经母细胞10，在两个4,6,8成神经细胞和神经胶质细胞和星形胶质细胞的钙波3受体通道表达的自发活动模式。由于在SVZ细胞是未成熟或有胶质属性，它们不火动作电位11,12，也就是说毫秒表示成熟的网络中的活动的电压电势的变化是不适用的在这个区域。因此，捕捉速度较慢的钙事件（以秒计）不仅是生物学意义的，但也许是最相关的...

<table border="1"><tbody><tr><td> 溶质 </td><td> 公司 </td><td> 目录编号 </td><td> 夹层（MM） </td></tr><tr><td>蔗糖</td><td>西格玛</td><td> S0389 </td><td>解剖:147毫米 学联:0毫</td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S9888 </td><td>解剖:42毫米 学联:126毫米</td></tr><tr><td>氯化钾</td><td>西格玛</td><td> P3911 </td><td>夹层:2.5毫米 学联:2.5毫米</td></tr><tr><td>氯化镁2 .6 H 2 O </td><td>西格玛</td><td> M9272 </td><td>夹层:4.33毫米 学联:1毫米</td></tr><tr><td>的NaH 2 PO 4·H 2 O </td><td>西格玛</td><td> S8282 </td><td>夹层:1.25毫米 学联:1.25毫</td></tr><tr><td>葡萄糖</td><td>西格玛</td><td> G8270 </td><td>狄氏ction:10mM的 学联:10毫米</td></tr><tr><td> 碳酸氢钠 </td><td>西格玛</td><td> S6014 </td><td>解剖:26毫米 学联:26毫米</td></tr><tr><td>氯化钙2 .2 H 2 O </td><td>西格玛</td><td> C3306 </td><td>解剖:1.33毫米 学联:2mM的</td></tr></tbody></table> 表1。夹层的解决方案和学联的化学品清单和食谱。 <table border="1"><tbody><tr><td></td><td></td><td></td><td> [头] </td></tr><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Vibratome </td><td>徕卡</td><td> VT 1000S </td><td></td></tr><tr><td>超能胶</td><td> Surehold或3M </td><td> 3G超级胶或3M兽医债券的Surehold </td><td></td></tr><tr><td>解剖工具</td><td> Roboz或特德·佩拉</td><td></td><td></td></tr><tr><td>氟钙离子敏感的染料</td><td> Invitrogen公司</td><td> F14201 </td><td></td></tr><tr><td>俄勒冈绿BAPTA-AM钙离子敏感的染料</td><td> Invitrogen公司</td><td> O6807 </td><td></td></tr><tr><td>的Pluronic F-127的20％的DMSO溶液</td><td> Invitrogen公司</td><td> P3000MP </td><td></td></tr><tr><td>立式激光共聚焦显微镜</td><td>奥林巴斯</td><td> FV300或FV1000 </td><td></td></tr><tr><td>浸水目标</td><td>奥林巴斯</td><td> LUMPlanFl 40×W / IR（NA 0.80）; LUMPlanFl 60×W / I（NA 0.90） </td><td></td></tr><tr><td>微操纵器</td><td>萨特</td><td> MPC-200/MPC-325/MPC-385 </td><td></td></tr><tr><td>压力控制器&lt;/ TD&gt; <td>派克汉尼汾</td><td> Picospritzer </td><td> &lt;3 PSI过程中的应用</td></tr><tr><td>用移液管拉马</td><td>萨特或Narshige </td><td>萨特P-97或Narshige PP-830 </td><td></td></tr><tr><td>玻璃吸液管</td><td>萨特</td><td> BF150-110-10 </td><td> ID:1.10，OD:1.50 </td></tr><tr><td>蠕动泵</td><td>哈佛设备</td><td>型号720 </td><td>流速:1毫升/分钟</td></tr><tr><td>商会浴</td><td>华纳仪器</td><td> RC-26 GLP </td><td>低调允许的客观间隙</td></tr><tr><td>管道</td><td>聚乙烯</td><td></td><td></td></tr><tr><td>温度控制器</td><td>华纳仪器</td><td> TC-324B/344B </td><td></td></tr></tbody></table> 表2。材料/设备清单。

preparation of acute subventricular zone slices for calcium imaging

脑室下区（SVZ）是在出生后的大脑的神经区域之一。 SVZ密密麻麻的细胞，包括神经祖细胞与星形胶质细胞的功能（SVZ星形胶质细胞），神经母细胞和中间祖细胞。出生在SVZ的神经母细胞切向迁移到嗅球，它们分化成的interneurons很大的距离。细胞间粘附分子和扩散信号的信号通过发挥着重要的作用，在控制神经发生。许多这些信号触发内和细胞间的信息发送的胞内钙离子活性。钙活性因此反射的活性的胞外信号，是一个最佳的方式来理解SVZ的细胞的功能之间的细胞间信号。 其他许多地区和类型的细胞，包括成熟的星形胶质细胞和神经元钙活动进行了研究。然而，传统的方法来加载，D细胞与钙指示剂染料（ 即浴加载）的效率不高在加载所有SVZ的细胞类型。事实上，细胞密度在SVZ排除内部的组织的染料扩散。此外，制备矢状切片，将更好地保留SVZ的细胞，特别是流延髓尾轴上的成神经细胞迁移的三维配置的。 在这里，我们描述的方法来准备矢状部分包含SVZ，装载的SV​​Z细胞与钙离子指示剂染料，收购活性钙与时间推移电影。我们使用荧光凌晨4点染料加载SVZ星形胶质细胞内组织施加压力。使用扫描共聚焦显微镜，让一个精确的分辨率为区分单个细胞钙活动的记录。我们的方法是适用于包括成人海马颗粒下层和胚胎神经源性区域的其他神经性区。此外，其他类型的染料可以使用所描述的方法被应用。

Watch this Scientific Journal Video about Preparation of Acute Subventricular Zone Slices for Calcium Imaging at JoVE.com

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

一个方法来加载脑室下区（SVZ）细胞中的钙离子指示剂染料记录活性钙。产后SVZ包括神经祖细胞和神经母细胞的细胞排列紧密。而不是使用清洗负载注入染料内的组织可以更好的染料扩散的压力。

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 ...

preparation-of-acute-subventricular-zone-slices-for-calcium-imaging

Research

JoVE Journal

Neuroscience

13.1K 次观看.  Yale University School of Medicine. 一个方法来加载脑室下区（SVZ）细胞中的钙离子指示剂染料记录活性钙。产后SVZ包括神经祖细胞和神经母细胞的细胞排列紧密。而不是使用清洗负载注入染料内的组织可以更好的染料扩散的压力。

视频: Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

脑室下区恩的脸：Wholemount染色和室管膜流量

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

科研

神经科学

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

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. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

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. ...

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 ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

新生儿脑室下区电穿孔

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...