这项工作是由英国医学研究理事会资助的项目为G0601253 GSB和RWB和SULSA Bioskape赠款，以资助GSB

这些方法是在在银行报告的研究中，RW 等 2小鼠入道通过颈脱位安乐死使用。在英国，这是在动物（科学程序）中列出的经依法批准附表1法法案，1986年，欧盟指令六十三分之二千零十/ EU。该联邦立法在香港仔的动物福利大学和伦理审查委员会谁批准的所有程序在本地执行。 1.准备耳朵标签<ol><li>制备标准生理盐水，如Liley的（1956）5，并用90％ 的 O 2/5％CO 2（卡波金）饱和它。 Liley的盐水是（mM计）: 碳酸氢钠 （12），氯化钾（4），KH 2 PO 4（1），氯化钠（138.8），MgCl 2的（1）， 氯化钙 （2）和葡萄糖（11）。注:在这个手稿的其余部分，"盐"指的生理盐水是与卡波完全饱和。在dissectioN，刷新覆盖每15准备生理盐水 - 20分钟。 </li><li>人道安乐死成年小鼠不会损坏头骨。注意:我们已经使用的C57 / BL6J和MF1小鼠，但可使用任何小鼠品系。最重要的方面是不使用大的成人（&gt; 25克）。标签在较大的老鼠就是不可靠，往往只局限于毛囊的小而散团。 </li><li>同〜25厘米剪刀取出外耳（耳廓）基地附近，只是头发浓密线以上，并在盐水淹没在硅橡胶衬里文化/培养皿（50毫米通常是方便）。 </li><li>耳廓前部（凹）朝下位置，并用细昆虫针定期针边距以硅胶（〜6 - 8穗，0.2〜毫米直径针）。刷新盐水，每15 - 20分钟，或者使用一个不断灌注浴。 </li><li>小心剥离由钝性分离前的皮肤后的皮肤，最初从耳廓上切基地访问厚厚的中央软骨和系统地对薄，无软骨的外部边缘工作。 <ol><li>要做到这一点，保持后皮肤的切割边缘的中心具有一对没有。 3钳。然后，用卡爪关闭，放置另一组没有的点。 3镊子在皮肤和底层软骨之间的差距。 </li><li>轻轻地从一侧到另一侧的间隙内的封闭镊子的点，使用所需的最小力，小心松开粘连而剥离背面的后部皮肤分层。 </li></ol></li><li>随着后皮去掉，留在位置前的皮肤。针后皮肤，皮肤的一面朝上，前皮肤旁（按照1.5以上）。 </li><li>其次，仔细，彻底去除夹在皮肤层之间由前皮肤由同一钝性分离技术，因为在1.6耳廓软骨。避免穿刺孔的镊子。 </li><li>然后中，f或者两者耳廓皮肤准备轻轻剥离，勇气和擦远结缔组织的薄层，类似膨胀聚苯乙烯泡沫，覆盖毛囊基地。注:获得最佳的结算可以采取一些做法;组织去除不足妨碍访问，无论对于染料溶液及用于成像，而刮太大力删除神经网络，它们在毛囊的底部。 </li><li>刷新生理盐水。 </li></ol> 2.披针形末端标记注:以下协议是苯乙烯吡啶染料进行了优化。另外，化学性质类似，苯乙烯吡啶染料应该工作，也许在浓度和培养时间有些调整后。处理染料溶液时，戴上手套和白大褂。无论是直接从厂家购买了10毫米股票染料溶液或生理盐水染料粉末溶解不含葡萄糖准备库存。分装（10微升通常是方便，但这种调整对于大SCA勒实验），并存储冷冻在-20℃可达6个月或〜1年-80℃。粉将保存至少2年。避免反复冻融/染料溶液的解冻周期;这一快速的变性染料。一旦解冻，储存在4°C和1周内使用。 <ol><li>预加热的水浴中30℃，用一台〜3 - 第5毫米水表面以下。 </li><li>新鲜制备10μM的苯乙烯基吡啶鎓染料溶液（10微升新鲜解冻的10mM苯乙烯吡啶鎓染料的10ml盐水），并在水浴平衡。 </li><li>销在硅酮橡胶各制剂内衬在1浸没35毫米培养皿 - 盐水2毫米（典型体积，〜2毫升）中。每个耳廓皮肤准备将产生两种制剂，如果削减了一半，从先端具小剪刀立足（10 - 14厘米长），动物尽量少用。 </li><li>将包含在30℃水浴中淹没平台上的盐水覆盖耳廓准备35毫米菜。确保水深度足以充分变暖，但在打开的盘不污染盐水。 </li><li>细脚（0.5 - 1毫米外径）管与流O 2 / CO 2到每道菜不断气制剂的有机硅基地。也放入水浴100ml为了清洗/菜清爽无染料的生理盐水。用塞子塞紧瓶保持卡波饱和度和防止水体污染。 </li><li>平衡在30℃30分钟，然后一切都更换过的盐水从100毫升塞的瓶中耳廓准备。孵育另外30分钟。注:此替代补偿盐水蒸发和烟气在线起泡造成的任何堆积体积损失。 </li><li>倒掉无染料盐水到100ml废物烧杯中，然后快速并小心吸干远离其余周围用纸巾制备的任何液体，以减少接下来要加入的染料溶液稀释效应。注意不要碰（ <em&gt;即损坏）暴露的深层真皮表面直接。 </li><li>在30℃的C 3含10μM苯乙烯吡啶鎓染料进行40分钟，然后返回到R / T的程序的其余部分 - 孵育耳廓制剂2。 </li><li>在三个阶段除去非内化染料，在R / T用的解决方案。注意:这些步骤在最小的环境光（遮光窗帘，红/橙低强度照明）表现最好，开始眼睛的暗适应成像。 <ol><li>倒掉从菜品的标签解决方案进入废物烧杯中，在快速连续无染料生理盐水三个转变迅速冲洗。这消除了残留苯乙烯吡啶染料溶液秉承的筹备工作，很容易从裸露膜departitions任何染料。 </li><li>在无染料盐水的终改变孵育另外30分钟，以从表面的膜departition最多剩余染料， 即染料不是由终端内化。 </li><li>删除持久染料卡住Ť直径:通过用磺化-b-环糊精衍生物螯合暴露的膜的外小叶（Advasep-7，1毫米，5分钟- 见表1）。因此，所有剩余的染料将在组织内。 </li></ol></li><li>在成像新鲜的无染料生理盐水将擦干的菜之外用纸巾彻底。 注:该终端现在已经准备好进行成像。要知道，披针形的结局是活的，因此，再慢慢释放的内吞苯乙烯吡啶染料是很重要的。尽管这将在R / T慢，它在成像期间以最小化/之前的延迟是重要的。如果实验需要多个制剂的标签，初始维持它们全部在30℃下未标记。它们将是可行的数小时。然后间隔依次标记，通过标记第二制剂（步骤2.7 - 2.8.3），而成像第一。 </li></ol> 3.与成像披针形结局。 注:观察员应暗适应以下成像步骤。增加视力这提供意味着较低的激发光水平是必需的，以找到用于成像的卵泡。这是用于最小化光毒性，而不是减少漂白的不便最重要的。通过全亮度照明产生的自由基能迅速（60秒内）杀死活神经末梢（GS比伊克，S和法杜勒WJ贝茨，未发表意见）。 <ol><li>成像之前，请检查解剖立体显微镜下的准备，小心地取出任何表面的碎屑。这防止自动荧光污染图像。 <ol><li>安装一个标准的荧光过滤器设置在一个直立的落射荧光显微镜阶段的菜（480 - 488 nm激发，500 - 520 nm发射的苯乙烯吡啶染料）。 </li></ol></li><li>减弱与中性密度滤镜的激发光强度，直至恰好足以从容定位终端秒。 </li><li>通过观察低标定位毛囊（3.5倍 - 4倍干燥目标），然后逐渐提高（10倍20倍和水浸泡的目的）的放大倍率的目标。 </li><li>通过对低功耗和高放大倍率20倍水浸泡的目标10X干客观捕捉标记披针形端子的图像。尽量减少光照强度和照射时间（0.5的一体化摄像机的时间 - 1秒建议）。 </li><li>一个标准的数码相机拍摄图像和使用专有软件保存计算机硬盘驱动器上的图像。注意:一个典型的例子示于图1。 </li><li>图片〜从各个准备20披针形的结局。在开始从切割边缘在耳廓皮肤和工作沿着这缘成像依次在每个毛囊底部的保证金最远。在稍微重叠的领域平行切割边缘，小心不要像同一毛囊加倍努力跨越任何被明显破坏。注意:再是典型的太多披针形终端形象它们都显著自发去染色发生之前。因此，我们建议您使用以下协议系统抽样人口。 </li><li>在完成边缘带之后，将一个场宽度朝向耳廓的基础上，并在相反方向重复该过程。 </li><li>像所有的终端遇到的，除了偶尔的人显然很导向更倾斜于光学平面，不大可能标准环形分析ROI内完全符合（见第4节）。不排除比周围端子，除非它们是明显损坏或背景强度特别高，指示局部组织损伤异常亮或更暗lanceolates。 </li><li>丢弃准备如果皮肤面积的10％以上/终端损坏/不可用。注意:作为lanceolates随时间脱色，完成成像洗涤的端部的20分钟内的每个制剂。 </li><li>一世F使用一个以上的准备，确保强度的比较都是有效的通过时间匹配的准备工作。要做到这一点，抵消〜30分钟染色各项准备，确保脱色斑次可比性计时。 </li><li>为了监测脱色斑（胞吐），图像在同一终端在5或10分钟的间隔。与实践中，它是可能的图象最多在任一制备每个时间点3个独立的端子。 </li></ol> 4.分析披针形标记结局。 注:以下步骤是分析终端强度的快速的方法。 <ol><li>使感兴趣区域（ROI）的一个环形区域在毛囊的底部（ 图2）为中心在毛发杆的尖端。设置环状的ROI足够大，以避免在任何毛囊毛干自发荧光的内周进行分析，但仍包括所有终端荧光。确保外周大到足以包围最大TERMIN人可能遇到的是面向靠近主要的光/卵泡轴。 </li><li>作背景的ROI面积大致等于目标环形（正方形或圆形，典型地〜400微米2），以确定在所分析的终端附近的本地染色强度。 注意:这些两个ROI的大小被设定在分析的开始和用来分析在所有的准备工作的所有后续卵泡/披端子中的任何一项实验。所以，在开始时小心以设置它们的参数是重要的。 </li><li>将背景的投资回报率足够接近毛囊代表背后的终端，没有毛囊之间的低强度皮肤皮脂腺当地背景，但要小心，不要包括任何末端区域。 </li><li>记录使用"措施"或"分析投资回报率的软件和数据传输到电子表格函数的两个ROI的平均亮度。</li><li>在电子表格中，对于每个单独的毛囊从减去平均本地背景强度的环形，得到净强度。这种调整对本地化背景大大降低了毛囊间差异。 </li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+endocytosis+in+outer+hair+cells+of+the+mammalian+cochlea.">Apical endocytosis in outer hair cells of the mammalian cochlea.</a> Eur. J. Neurosci. 20 (1), 41-50 (2004).</li><li>Watson, S., Aryiku, C., Banks, R. W., Bewick, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+gadolinium+and+FM1-43+as+blockers+of+stretch-evoked+firing+of+rat+muscle+spindle+afferents.">Comparison of gadolinium and FM1-43 as blockers of stretch-evoked firing of rat muscle spindle afferents.</a> Proc. Physiol. Soc. 21, P22 (2010).</li></ol>

作者什么都没有透露。

比伊克和贝兹最初开发的N-（3- triethylammoniumpropyl）-4-（4-（二丁基氨基）苯乙烯基）吡啶鎓溴化物相关苯乙烯吡啶鎓染料7-10来监测突触小泡膜的当地的回收。染料摄取，并因此增加了荧光，因此反映了突触小泡膜的内化（内吞作用）。随后，这个内化染料可以通过进一步的电活动重新释放，显示出染料损失监视囊泡膜外化（胞吐作用）。在突触，这是对神经递质分泌的代理。与此同时，我们也发现这些染料标记的主mechanosensory神经末梢7。最近11，比伊克，银行和同事然后表明，这反映了成熟，分化mechanosensory端子体外一个类似的过程。这里再一次，该染料标记50纳米直径"突触样&#39;该回收组成释放谷氨酸囊泡（SLVS）。在mechanosensory终端，不像他们的突触同行，这个循环主要是自发的。它也是通过机械的，而不是简单地电，刺激调制。 在培养和在耳蜗毛细胞，特别是在一般和苯乙烯基吡啶鎓染料苯乙烯基染料的感觉神经元已显示通过mechanosensory通道，阻断mechanosensory频道和不可逆地标记内部的膜12。然而，在原位分化mechanosensory终端，这里可比苯乙烯基染料浓度在披针末梢2，或在IA神经末梢中肌梭11，并在没有被机械刺激13,14毛细胞，标签是由膜的内吞作用。在mechanosensory神经末梢，例如，标签是可逆的，并且不会阻塞在这里使用2，11，1中的浓度mechanosensory响应5。尽管在这些神经末梢通道渗透一些染料的内化，不能完全排除与latrotoxin近总脱色指示大多数成熟终端标记的是通过与再循环小泡膜的内化。我们因此，通过检查对染料的吸收和释放药物效果时使用此技术来研究活动依赖性11，以及最近，SLV回收在mechanosensory传入端子的药理2。 与大多数实用技术，重现需要重复和实践。一些用于确保可靠性的关键点现在将讨论。在较年轻的动物披标签是更可靠 - 我们已经使用了最小的动物为15克体重。它是不完全清楚为什么是这种情况，但它可能是因为年轻的结缔组织的解剖需要较少的机械损伤和乐鸟类去除组织覆层神经分布较少时，剩余的残渣。 总体而言，组织制剂是相当简单的，只用在老年小鼠中剥离表层分开是最技术挑战性应用方面，然后。在这些中，表层在基底附着强烈一起解剖开始的地方，但即使在各层的分离变得朝向边缘容易得多。值得庆幸的是，或许因此，这些边缘较薄的皮肤区域通常给出最完满的标签，因为一般不前的皮肤。因此，特别是避免抓的利润率非常薄的皮肤。为了最大限度地减少损坏的风险，通过​​用封闭镊子直接推压，而不是抓间接操纵的制剂，或者，如果必要把握不可能只强硬组织至被标记。在去除"聚苯板"层正上方毛囊彻底，因为这是我的主械阻碍成像和药物的访问。然而，有必要以略低于底层结构，因为这损害（ 即除掉）的神经网络和终端的物理接触最小化。如果主要的荧光黄/白的皮脂腺，毛干基地和橙色/黄色披针形结局几新月突出自体荧光，这表明神经丛层和相关披针形端子通关过程中已被删除。这似乎是与旧（&gt; 30克）的小鼠的主要问题。整个染色过程，确保一切是在30℃和良好的氧化。请注意，标记终端还活着。因此，染料会被持续的自发（构）胞吐释放分泌。这将是在R / T慢，但它是很好的做法，以尽量减少去除螯合剂的解决方案和成像之间的延迟，以减少染料损失。此外，对于间的基团比较小号强度的案例研究，这是至关重要的，以确保所有的组的成像时间匹配。成像前的染料螯合剂使用极大地提高了图像对比度。所以，虽然比较昂贵，螯合步骤是确保良好的图像质量非常重要。螯合剂的使用可以通过多次重复使用的溶液被最小化，甚至在2 - 连续3天，如果使用之间储存在4℃。丢弃螯合剂解决方案，一旦它进入明显的粉红色，可见其染料封存已接近饱和。 在成像过程中，应注意对这些活端子光毒损伤，这是强度和激发光的照射时间两者的累积的结果的潜力。这考虑是单一的时间点映像，请在图像质量是首要关注的问题更重要。然而，这是在反应动力学研究反复成像期间至关重要激励光照射降低到最低限度。尽管开发这些染料标签突触，我们发现激发了满功率激发照明&gt; 1分钟将导致神经末梢戏剧性和不可弥补的损害。他们首先随后扩大巨大的，然后过了一段约15分钟完全崩溃。我们没有系统地测试曝光时间和强度的相对贡献，但这些意见建议的指导原则应该是最大限度地缩小了。这样，它建议在激励光强度和持续时间的最小与得到良好图像相称，并且适当地控制图像的拍摄在时间过程研究。要测试的数据不重复的成像条件下受光毒性，在每个时间点取一个先前未观看终端的附加图像。这是为了确保在幼稚终端标记的行为类似于在卵泡被反复成像。 许多因素可降低组内变化的净强度D的ATA。的ROI的准确位置，特别是背景的ROI，是很重要的，因为即使是很小的不适当染色的区域可以极大地影响毛囊间的数据的变异性。净强度的变化也被完全怎么每个毛囊由结局栅栏包围的影响。例如比较，在图1B中的月牙形标记分布与图2所示的几乎完全的圆形图案。更复杂的分析，可能使用自动化的软件检测和阈值，是必要的，如果包围的程度是一个相关的参数。请注意，即使是最准确分析卵泡基的强度分布不是正态分布的（ 见图3），因此有必要对人口中位数，而不是装置的治疗间比较使用非参数统计的。规范化技术，如表达强度为百分比ö˚F对侧控制，或对照组由几个制剂，可以适用于进一步减少可变性。要考虑的最后的技术项目为药理试验的实验设计。在药理研究，预孵育在药物溶液中的准备之前，染料应用30分钟。然后，保持在染料溶液中的药物浓度。对照，使用生理盐水或，如果药物被溶解在车辆中，盐水加车辆。 本文章演示了这种新的耳廓准备在皮肤最少的准备如何提供mechanosensory结局的高质量的图像。我们还表明它可用于检查的囊泡循环的两个关键方面药理学。我们首先表明，谷氨酸受体激动剂可以增加染料内在（内吞作用的标记物），以及第二，染料与latrotoxin（胞吐作用的兴奋剂）再次丢失。这preparat的意义离子技术，它提供给现场的成像实验住皮肤mechanosenory终端，获得独一无二的机会，终端功能，结构及其相互关系的光学监测的便利交通。对此末后，我们还开发了进一步对组合的光/电生理学研究（详见妹妹朱庇特的文章）使用。

Mechanosensory神经末梢一般较小，弥漫性分布，难以就地获得，因为它们通常埋在皮肤或其它周围组织深。可视化它们，因此，通常需要切片，随后通过一个延长的免疫标记/染色时期，或延迟和费用获得具有荧光标记，如绿色或黄色荧光蛋白（GFP / YFP）1的基因表达小鼠线。在这里，我们展示了方便组织和一个快速和简单的方法来获得大量毛囊传入的研究，以及他们如何可以快速标示端子功能2的光学监测。与实践中，从解剖成像整个技术可以在少至2小时内完成。 感觉轴突的披针形端子支配毛囊哺乳动物周围形成毛囊的上皮栅栏，每个终端夹胶质/雪旺细胞之间的处理2-4。端子的目的是检测它们包围的毛发的机械位移。它们是快速和缓慢适应结局的混合物，但它们主要产生活性的短脉冲响应于毛发的运动。通常，烧成停止运动停止时非常快，即使在持续位移的存在。 用于通过光学方法研究披针端子这样鼠耳廓模型对研究这些端部的结构和功能的许多有利的特征。耳廓是皮肤的主要两层并列背到背，只有之间的软骨组织的少量。皮肤是非常薄，容易解剖由于最小量相对于主体的其他区域弱粘合剂的结缔组织。在易分离皮肤层，因此，给毛囊和终端很方便。神经支配为方便和识别。毛囊更稀疏分布比其他皮肤区域，促进卵泡个人或小团体的摄像。薄底层真皮层提供良好的无障碍染料和药理药，所以是理想的未经进一步加工利用荧光显微镜成像。标终端的成像可以是活的终端，或者，如果使用的是可固定染料类似物，固定和进一步的组织学处理之后。 我们已经使用该制剂表明膜中的披针末梢发生回收，通过摄取（内吞作用）和苯乙烯基吡啶鎓染料（FM1-43的释放（胞吐作用）证明N - （3- triethylammoniumpropyl）-4-（4- （二丁基）苯乙烯基）二溴化吡啶）。该染料不，但是，似乎显著标记投资雪旺氏细胞处理2。我们还发现，这种染料吸收/释放，因此膜 - [Recycling，需通过一个非典型（磷脂酶D耦合）代谢型谷氨酸受体调节谷氨酸。简单的刺激和协议分析的结果都说明，共同潜力分析问题也突出。

<table border="0" cellpadding="0" cellspacing="0" width="1435">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PDMS - Sylgard 184</td>
			<td style="width:111px;">Dow Corning</td>
			<td style="width:119px;"></td>
			<td style="width:989px;">Flexible, inert, translucent solid silicone polymer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">No. 3 Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11231-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Austerlitz Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>Very fine pins to attach pinna preparation securely to the PDMS with minimal damage.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FM1-43/Synaptogreen C4</td>
			<td>Biotium/Cambridge Bioscience</td>
			<td>BT70020</td>
			<td>Fluorescent membrane probe that reversibly partitions into the outer leaflet of cell membranes. Used predominantly for monitoring vesicle membrane endo-/exocytosis.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Advasep 7</td>
			<td>Biotium/Cambridge Bioscience</td>
			<td>BT70029</td>
			<td>A sulfonated b-cyclodextrin derivative that chelates FM1-43 (&#38; other styryl pyridinium dyes) out of the exposed membranes, leaving internalised dye to be seen more clearly by lowering the background labelling/fluorescence.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retiga Exi Fast 1394</td>
			<td>Qimaging</td>
			<td></td>
			<td>Monochrome, cooled CCD camera - basic model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Volocity 3D Image Analysis Software</td>
			<td>Perkin Elmer</td>
			<td>Volocity 6.3</td>
			<td>Image capture and analysis software.</td>
		</tr>
	</tbody>
</table>

optical monitoring of living nerve terminal labeling in hair follicle lanceolate endings of the ex vivo mouse ear skin

被用于光学监测染色和包围毛囊在小鼠耳廓的皮肤披针形端子的脱染色描述了一种新的解剖和记录技术。制备简单，比较快的，可靠的高产生活神经末梢来研究囊泡循环的研究，广泛用于苯乙烯吡啶染料的吸收和释放的多重标记单位的广泛地区。标记前细分的准备工作使得测试主场迎战从单一个体来说都是一样的耳朵控制的比较。有用的技巧，给出了改进的制备，标记和成像参数的质量。这种新的系统适用于药理学测定和机械诱导的摄取和在野生型中披针形端子，这些活体染料和转基因动物的释放。上标记强度调节影响的实例中给出。

毛囊在此耳廓制备很容易下透射可见无荧光（ 图1A），示出了制备的极薄的性质和无障碍的相对容易它可以提供给这些mechanosensory端子。每个被突出的毛干基刺入皮脂腺的黑暗新月代表。下表面荧光，围绕每个毛囊标记的披针形端子被清楚地看到，并显示通常健壮自发苯乙烯基染料的吸收（ 图1B）。这种现象不会强加的运动。的披针形端子被视为以包围发干，虽然包围的程度是可变的6。许多标记是点状（点），而不是所预期的线性排列。该点状图案反映在光平面被观察沿着端子的长度的端子。氏■准备没有接收到进一步的处理可视化标记后，它被简单地转移到显微镜阶段和原位 ，这再次说明了这种制剂的简单成像。 实施例实验到这种新颖的制剂是适合于图3中示出，它可以被用来研究两个内吞和胞吐生活终端，以及它们的调制。因此，对于内吞作用，谷氨酸盐受体激动剂可以增加标记强度，同时与配体或阳离子，例如Mg 2+阻断钙通道，镍离子或Cd 2+减小它2。可替代地，该制剂还可以用于研究胞吐的动力学， 如图3C所示 。首先，标记终端自发地看出脱染色。然而，这种脱色由胞吐兴奋剂加快，latrotoxin。更多德实验结果的尾巴可以在银行中可以看出，RW 等人，2。 <img alt="图1" src="/files/ftp_upload/53855/53855fig1.jpg" /> 图1.强大的苯乙烯基染料标记记录在小鼠耳廓 。 （ 一 ）明视场照明未标记的耳廓准备工作的一部分，显示出毛囊如何清楚地出现在清除覆乳晕/脂肪组织的面积。黑暗中，通常bilobed，月牙形状围绕中央竖毛皮脂腺。 （B）类似的区域，以略高的放大倍率和落射荧光成像，显示出标有苯乙烯基染料披针形的结局。比例尺- ​​40微米<a href="https://www.jove.com/files/ftp_upload/53855/53855fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> "1"&gt; <img alt="图2" src="/files/ftp_upload/53855/53855fig2.jpg" /> 图2.分析苯乙烯基染料标记强度 。 （A）的毛囊标记与苯乙烯基染料的典型圆形图案，集中于毛干（该图像中不可见）。 （ 二 ）主要分析"感兴趣区域"（ROI）被叠加到披针形神经末梢包围毛囊的栅栏的位置，一个标准的定义环。这个投资回报率保持在形状和面积恒定的任何单个实验，以确保标签的强度可以制剂间和跨所有的治疗得到公平的比较。每个ROI的净强度减去相邻的方形或圆形的背景区域（〜400微米2）的强度来计算。同样，这个广场的背景区域遍及任何给定的实验中保持形状和面积不变。目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53855/53855fig3.jpg" /> 图3.毛囊传入Lanceloate结局的标记强度不呈正态分布，并可以用来检查这两个内切和胞吐作用。从苯乙烯染料强度测量典型数据使用上述分析方法自发标识披针形端子。 （ 一 ）控制的条件下，标记强度（54卵泡，4耳）和1mM谷氨酸（42卵泡，4耳）。个别结局的强度值显示为集群散点图。每个点代表围绕一个卵泡一个终端栅栏的强度。请注意，即使在控制条件下的几个数据点（毛囊）是非常激烈的，而大多数是在较低强度聚集。在特定INTENS所述第一点性是在中心绘制和相同强度的随后的数据点逐渐多个横向绘制在两侧。因此，横向扩散代表任何特定标记强度的卵泡的频率。水平线表示平均±25％四分位数间距。孵育1 mM的谷氨酸由〜50％（*** P &lt;0.001，曼 - 惠特尼）增强了中位数强度，但标签可以通过200增强 - 300％，在一些终端。 （B）图片展示谷氨酸介导的增强型染料摄取（胞吞作用）。谷氨酸（1毫米）的毛囊制剂孵育显着提高苯乙烯基染料的吸收，如由增加的标记强度。比例尺为20μm。 （ 三 ）加强染料释放（胞吐）。标记后，苯乙烯基染料自发再次释放，如在0分钟的标记比在60分钟显然更亮，甚至背景扣除后，以控制光漂白。此版本是大大婆通过latrotoxin tentiated，黑寡妇蜘蛛的毒液成分，从而引发无法控制的囊泡胞吐。在毒素60分钟之后，末端标记几乎是检测不到的。苯乙烯基染料标记的这种可逆性支持的假设终端荧光突触小泡状的当地的回收过程中，由于内部化。比例尺20微米。 <a href="https://www.jove.com/files/ftp_upload/53855/53855fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin at JoVE.com

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

optical-monitoring-living-nerve-terminal-labeling-hair-follicle

Research

JoVE Journal

Neuroscience

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

7.4K 次观看.  University of Aberdeen. Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

视频: Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy 1. OHCs are able to change shape &#x2014;elongate, shorten and bend&#x2014; in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals 2.
OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization 2, 3. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) 4. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins 5, 6. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research 7.
The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) 8. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches 9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques 12. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. 
We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

调查外毛细胞运动与外部的交流电场刺激和高速图像分析相结合

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives ...

科研

神经科学

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

小鼠皮肤及其毛囊图案和感觉轴突形态分析中的应用平山成像

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

机械刺激传入输出和神经末梢标签联合录制在小鼠毛囊披针形结局

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

在毛囊披针形结局生活神经末端标记的光学监测

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