Name: physiological preparation of hair cells from the sacculus of the american bullfrog (rana catesbeiana)
Uploaded: 2017-03-17T15:00:00.000+00:00
Duration: 12 min 7 s
Description: Watch this Scientific Journal Video about Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana at JoVE.com

The authors wish to acknowledge Dr. A. J. Hudspeth for funding and expertise in developing the preparations described in this paper. We also wish to thank Brian Fabella for creating and maintaining much of the custom equipment and software used in this protocol.
J. B. A. is supported by grant F30DC014215, J. D. S. is supported by grant F30DC013468, and both J. B. A. and J. D. S. are supported by grant T32GM07739 from the National Institutes of Health.

伦理学声明:以上程序进行的机构动物护理和使用委员会（IACUC）在洛克菲勒大学的批准。 1.预实验Prespanparation <ol><li> 解决方案 <ol><li>准备丁香酚溶液（2.5克·L -1·kg -1时青蛙）。制备盐溶液（ 表1）。 注:对于一室准备，准备人工外淋巴液;对于双室准备，准备人工两者外淋巴和内淋巴人工。 </li></ol></li></ol> 2.实验工具<ol><li> 玻璃纤维刺激的制备 <ol><li>缩小与在单行拉具有高耐热和高流速的电极拉出器硼硅玻璃毛细管中。加载拉毛细管成螺线管驱动牵拉。 </li><li>使毛细管的尖端朝着长丝玻璃珠熔化到它，直到毛细管接触玻璃珠。 </li><li>打开灯丝到毛细管的尖端融进了玻璃珠。一旦毛细管和胎圈之间的玻璃形成薄薄的桥梁，从而关断灯丝和大约在同一时间激活电磁车夫。 注意:这将拉动玻璃毛细管成直角，以它的长轴，创造了坚实的光纤。 </li><li>确保纤维的直径不超过0.5 - 1微米，其长度不超过100 - 300微米。如果纤维的长度超过了这个尺寸，与虹膜剪修剪。使用已经沉闷，以避免损坏清扫工具剪刀。 </li><li>以提高光对比度，溅射涂层的光纤。使用溅射涂布机用金 - 钯源。每个光纤垂直加载到其对源锥形端一个溅射镀膜机。使纤维的前端为1的距离 - 2厘米的金 - 钯源。 </li><li>关上溅射镀膜机的腔形成密封，并打开它。反复冲洗出周围空气中氩气。冲洗后的空气，减少该腔室至10Pa（70毫托）的压力。 </li><li>溅射涂层在10秒脉冲，10秒延迟超过120个课程。 注:光纤的尖端就会变暗了本协议的期限，如果溅射镀膜是成功的。 </li></ol></li><li> 尖锐的微电极的制备 <ol><li>用一个线高热量拉内部长丝拉玻璃毛细管。当填充有3米氯化钾300兆欧 - 电极应具有100的阻力。装满3米氯化钾每个电极。 </li><li>弯曲每个电极的与microforge尖端安装在放大器探头16时，以使其在垂直于毛细胞顶端表面。 </li></ol></li><li> 离子导入移液器的制备 <ol><li>拉的玻璃毛细管具有内部灯丝50MΩ提示。用浓溶质（如 500毫米硫酸庆大霉素）填充。 </li></ol></li><li> 铝安装方的制备 <ol><li>切割铝箔的1厘米×1厘米见方。穿孔箔在与脑脊髓刺毁杆或其它尖锐物体的前端的中心。塑造穿孔，以便它是圆形的，直径约为1毫米。 </li></ol></li><li> 真空润滑脂填充注射器制剂。 <ol><li>删除一个5毫升的注射器柱塞。从真空润滑脂回填注射器。更换柱塞。 </li></ol></li><li> 聚四氟乙烯涂胶器的研制 <ol><li>做一个长2毫米的轴向切木涂药棒的末端。观看端上时，缝隙应在施用器的中心。 </li><li>用锋利的剪刀或刀片，切聚四氟乙烯2毫米×4mm的矩形从1mm厚的片材。 </l我&gt; <li>用剃刀刀片聚四氟乙烯矩形的2毫米长的边薄之一。在矩形的中间开始，通过切片向外以形成在矩形的前端的斜角减小矩形的厚度。 </li><li>将聚四氟乙烯矩形进木制涂药缝隙使得斜边面临来自棒了。应用5分钟环氧树脂聚四氟乙烯矩形固定到位的基础。允许涂胶器以固化在室温下1小时。 </li></ol></li><li> 安装下半部的两个腔室的制备 <ol><li>机或3D打印双室的下半部分安装（ 参考文件2）。确定腔室的下表面上的直径为20mm的凹圆。 </li><li>适用的环氧树脂的薄层到凹圆的横向最3毫米。在环氧树脂应用一名年满18 mm圆柱玻璃盖玻片。允许环氧树脂固化1H。 </li></ol></li></ol> 3.内耳器官的提取<ol><li>通过将其放置在含有丁香油酚麻醉液10分钟，小斗麻醉的美国牛蛙。调节溶液的体积，使青蛙的humero肩胛关节谎言只是液气界面的上方。 </li><li>安乐死双穿髓的麻醉牛蛙。 <ol><li>抓住青蛙麻醉用一个手指鼻子上面，另一个在下颌下方，并向前旋转青蛙的头。 </li><li>迅速大幅下挫穿髓杆插入通过枕骨大孔，这是在青蛙的枕部进程之间的中线发现颅顶。 </li><li>慢慢地退出，直至尖端从枕骨大孔离开旋转杆。尾部强制杆通过椎间孔椎体破坏脊髓。 </li><li>确认青蛙通过观察其下肢延长正确毁脊髓加倍。 </li></ol></li><li>抓住青蛙用拇指鼻子和食指抓犁骨齿，提高了稳定性之上。通过切断双侧颞下颌关节，随后垂直切割的rostrocaudal轴斩首青蛙。为了确保颅骨内的内耳器官保持完整，确保切尾是这两个鼓膜。 </li><li>使用stereodissection显微镜，执行通过从锄骨齿腭组织切成组织（ 图1A）的最后程度中线。 </li><li>断绝与手术刀水平削减任何肌肉躺在下面的腭组织透露后软骨清晰。除去肌肉后，观察时间软骨形成听囊的边界的棒棒糖形状。 </li><li>断绝其与点的耳囊软骨接触的鼻小柱。 </li><li>通过反复shallo这刮胡子软骨薄层通过它W型卧式削减。避免大幅削减，以防止对内耳器官造成伤害。这将打开时间软骨（ 图1B）的棒棒糖结构中找到的听囊。内听囊是青蛙的内耳器官（ 图1C）。 </li><li>修剪后和听囊的侧边，小心不要损坏内耳器官。在夹层，经常流过盐水的内耳器官，以确保它们仍然淹没和水分。 </li><li>定位在软骨到中线的内侧连接在颞骨的两个圆形开口。通过最内侧向下开口切破裂颞骨。 </li><li>进行第二次向下通过削减在其postero外侧边听囊中。取出片这和以前的切口之间的软骨，以提供对内耳器官。 </li><li>从撬3.8的步骤一个松散的切换之间的软骨ND 3.9和耳胶囊剪了。这个动作切断最近半规管，然后可以用作用于进一步操纵的手柄。 </li><li>注意不要触碰小囊，切断第八次颅神经。 </li><li>保持最近的半规管壶腹。轻轻转动内耳揭露剩下的两个半规管。一旦暴露每一个管道，它断绝。 </li><li>握住神经或半规管壶腹，从头部提取内耳，并将其放置在一个充满冷充氧人工外淋巴液一道菜。内耳的除去允许颞骨，通过该半规管一次传递（ 图1D）内的通道的可视化。 注:重复上述步骤，提取第二耳。 </li></ol> 4.一室准备<ol><li> 球囊的分离 <ol><li>通过识别其庞大丝毫找到球囊耳石电子的质量和摆在它之上的囊状神经（ 图1E）。小心不要为了保持囊状毛细胞的完整性以下步骤中物理精神创伤小囊。 </li><li>修剪半规管来呈现内耳的机动性。 </li><li>拆下水箱外淋巴覆盖球囊的神经侧面。使周围的蓄水池周边温柔的削减。接着切割组织的小柱子是弥合池的膜小囊的神经侧面。 </li><li>除去lagena及其相关的神经。 </li><li>握住囊状神经，轻轻提起球囊，并通过otoconial囊薄膜切割。由于耳石洒出囊，通过围绕其周边切割释放球囊。 注:分离该小囊后，剩余的内耳器官如果需要，可以保存。所述球囊的除去减轻其他结构，例如作为识别两栖动物和基底乳头。 </li><li>用剪刀或镊子小心地擦去剩余的球囊任何耳石。请注意，不要在此过程中接触到球囊。 </li><li>剪掉球囊"边缘的任何剩余otoconial囊膜。该膜趋向于附着到塑料和玻璃的表面和它的去除最小化在处理组织的挑战。 </li><li>轻轻流盐水在球囊用巴氏吸管以除去任何残留耳石（ 图1F）。 注:重复此过程对第二球囊，它是第一个镜像。 </li></ol></li><li> 消化和安装 <ol><li>使用巴斯德吸管以其尖折断的后端，分离的sacculi转移到含有3毫升67毫克∙L-1蛋白酶XXIV的在人工外淋巴培养皿。 注:此蛋白酶处理消化圈养每个kinociliary灯泡的耳石链接膜，使膜去除而不损坏感觉毛束。 </li><li>温育30分钟将组织在22℃（或在21℃35分钟）。每个消化球囊转移至一个开口面的实验室，并与磁销固定组织。 </li><li>囊状黄斑直接位于下方的耳石膜是包含毛细胞的球囊的一部分。识别它，小心地取出用细睫毛上覆耳石膜，小心不要碰到囊状黄斑。 </li></ol></li></ol> 5.双室准备<ol><li> 球囊的分离 <ol><li>隔离从内耳器官球囊在4.1节。 </li></ol></li><li> 安装和消化 <ol><li>填充人工外淋巴液安装块（ 参考文件3），并把穿孔铝箔上的一个开口，利用真空的两个点油脂举行的地方，形成一个弱密封。 </li><li>传送一个球囊到箔和居中上朝下黄斑和朝上神经断端孔的顶部。 </li><li>删除周围用一块扭曲的组织的球囊生理盐水。灯芯盐水干燥环绕球囊的铝方的表面上。 </li><li>使用聚四氟乙烯涂布器施加氰基丙烯酸酯胶，以形成沿球囊的边缘和铝方之间的边界处的密封。确保该球囊的整个圆周被覆盖有胶。 注意:出发太慢允许氰基丙烯酸酯胶蠕变在球囊的神经侧面，并最终覆盖。因此，当务之急是迅速完成此任务。 </li><li>放置盐水上所安装的组织的顶部下降到固化的粘合剂。胶的薄膜可以形成于盐水的掉落顶部;用镊子将其删除。 </li><li>小心地除去叔他从安装块箔。翻转上的组织，以使球囊的黄斑一侧朝上，浮它在人工外淋巴液充满培养皿。 </li><li>添加对黄斑顶部的蛋白酶XXIV溶液一滴和在22℃（或在21℃35分钟）温育30分钟。填充双室装置（ 参考文件2）与周围的中央室外淋巴和地点真空润滑脂的下管。 </li><li>将铝箔装在小囊，其神经面对容器的表面下室。添加油脂围绕箔片的周边。 </li><li>放置在箔制备的上部腔室（ 参考文件1）中 ，小心，以形成与真空润滑脂一个完整的密封。装满冒泡人工淋巴上腔，并轻轻地用睫毛去除耳石膜。 </li></ol></li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+active+motor+model+for+adaptation+by+vertebrate+hair+cells.">An active motor model for adaptation by vertebrate hair cells.</a> Journal of Neuroscience. 12 (9), 3291-3309 (1992).</li><li>Crawford, A. C., Evans, M. G., Fettiplace, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+adaptation+of+transducer+currents+in+turtle+hair+cells.">Activation and adaptation of transducer currents in turtle hair cells.</a> The Journal of physiology. , 405-434 (1989).</li><li>Howard, J., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+relaxation+of+the+hair+bundle+mediates+adaptation+in+mechanoelectrical+transduction+by+the+bullfrog's+saccular+hair+cell.">Mechanical relaxation of the hair bundle mediates adaptation in mechanoelectrical transduction by the bullfrog's saccular hair cell.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (9), 3064-3068 (1987).</li><li>Purves, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+release+of+drugs+from+iontophoretic+pipettes.">The release of drugs from iontophoretic pipettes.</a> Journal of Theoretical Biology. 66 (4), 789-798 (1977).</li><li>Tinevez, J. Y., J&#252;licher, F., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unifying+the+various+incarnations+of+active+hair-bundle+motility+by+the+vertebrate+hair+cell.">Unifying the various incarnations of active hair-bundle motility by the vertebrate hair cell.</a> Biophysical journal. 93 (11), 4053-4067 (2007).</li><li>&#211; Maoil&#233;idigh, D., Nicola, E. M., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diverse+effects+of+mechanical+loading+on+active+hair+bundles.">The diverse effects of mechanical loading on active hair bundles.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (6), 1943-1948 (2012).</li><li>Kennedy, H. J., Crawford, A. C., Fettiplace, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+generation+by+mammalian+hair+bundles+supports+a+role+in+cochlear+amplification.">Force generation by mammalian hair bundles supports a role in cochlear amplification.</a> Nature. 433 (7028), 880-883 (2005).</li><li>van Netten, S. M., Khanna, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffness+changes+of+the+cupula+associated+with+the+mechanics+of+hair+cells+in+the+fish+lateral+line.">Stiffness changes of the cupula associated with the mechanics of hair cells in the fish lateral line.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (4), 1549-1553 (1994).</li><li>Bormuth, V., Barral, J., Joanny, J. F., J&#252;licher, F., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+channels'+gating+can+control+friction+on+vibrating+hair-cell+bundles+in+the+ear.">Transduction channels' gating can control friction on vibrating hair-cell bundles in the ear.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (20), 7185-7190 (2014).</li><li>Crawford, A. C., Evans, M. G., Fettiplace, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actions+of+calcium+on+the+mechano-electrical+transducer+current+of+turtle+hair+cells.">The actions of calcium on the mechano-electrical transducer current of turtle hair cells.</a> The Journal of physiology. , 369-398 (1991).</li><li>Kozlov, A. S., Risler, T., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+motion+of+stereocilia+assures+the+concerted+gating+of+hair-cell+transduction+channels.">Coherent motion of stereocilia assures the concerted gating of hair-cell transduction channels.</a> Nature Neuroscience. 10 (1), 87-92 (2007).</li><li>Lumpkin, E. A., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+free+Ca2++concentration+in+hair-cell+stereocilia.">Regulation of free Ca2+ concentration in hair-cell stereocilia.</a> Journal of Neuroscience. 18 (16), 6300-6318 (1998).</li></ol>

The authors declare no competing interests.

在牛蛙的球囊骗几千易于访问的感觉毛细胞。这里，我们证明提取和准备球囊为单组分和双室录音。这两种制剂使毛细胞及其相关的束微机械都和电生理研究。由于组织可以与频繁更换充氧的生理盐水几个小时生存，实验可能会持续较长的时间。在这些制剂中毛细胞通常为微电极记录解剖后长达6小时保持存活，而发束振荡自发进行萃取后长达24小时。 小囊的成功提取和安装有赖于克服一些共同的挑战。首先，在囊状黄斑的顶表面直接接触应在整个制备过程来避免。囊状神经提供了安全manipu方便的提手小囊的相关特征研。一旦从所述内耳器官的其余部分释放，所述球囊应采用大口径吸管而其余浸入流体以避免其感觉上皮机械损伤进行传输。耳石从黄斑表面除去必须无毛细胞机械损伤完成。因为耳石直接躺在上面斑，毛发细胞可以通过清扫工具，并在除去耳石的耳石膜之间的意外接触而损坏。为了避免损坏，我们建议耳石的凝胶状物质被在位置远离黄斑保持并作为单质除去。这避免了otoconial质量碎裂成无数集群，其中每个将被单独提取。如果耳石的小簇保持它们可以与由巴斯德吸管输送柔和流体压力被除去。最后一个挑战涉及密封在小囊和铝安装方之间形成双室制备。采用具有穿孔足够小以允许的球囊和周围铝许可证的组织的完全密封之间约100μm的重叠的正方形。胶应，以便形成紧密的密封可接触与约100微米左右的黄斑的周边囊状组织。 游离的Ca 2+浓度是在毛细胞研究中的一个重要考虑因素。 的 Ca 2+调节快速和缓慢适应，从而确定机械传导装置的动力学和的发束的有源处理现象的特征，包括自发束运动8,23。 体内钙的膜迷路积水出现在250微米，因此最生理有关动力学在此浓度（茂盛JHR，R.雅各布和AJ Hudspeth评估。UnpublisHED观察16）。然而，从毛细胞微电极记录需要外部的钙浓度超过2毫米左右的微细胞膜的正常密封。因此，必须使用高钙生理盐水这些实验。最后，我们不妨来研究细胞外钙的在机械传导采用多种钙离子浓度的影响。在这些情况下，重要的是记住低于1μM钙浓度通常会导致末梢链路破裂和转导28的不可逆损失是很重要的。 这里所描述的两个实验准备允许的范围内对毛细胞生物物理测量。然而，额外的测量可以用稍微修改这些制剂制成。在折叠囊状制剂，头发束横向可视化。从这样的高度成像头发束运动揭示连贯MOT短期和高大的纤毛29的离子。在这里，囊状黄斑首先从它的底层组织中分离，随后沿着囊状神经，使发束朝外，并在折痕横向可视化定义的轴折叠。第二个修改，毛细胞分离，使双方的毛细胞的软件包及其胞体的研究。毛细胞机械分离到用于成像和电生理记录30载玻片。最后，毛细胞可以从上皮通过遵循类似的解离协议，但没有机械解离步骤挤出。该处理导致毛细胞逐渐挤压出来的上皮，提供电生理记录基底访问，同时尽量减少机械损伤。这些制剂和它们的许多修改演示青蛙球囊的通用性，作为生物物理的模型系统感觉毛细胞的研究。

哺乳动物的acousticolateralis器官具有一个复杂的结构和位于解剖利基，可以是难以进入的范围内。例如，哺乳动物耳蜗包括一个螺旋迷宫和嵌入在厚颞骨内。耳蜗的隔离常常导致位于其内的感觉细胞的机械损伤，因此，已被证明是一项困难的任务1。因而神经科学家已经转向模型，更容易从耳朵的密室提取系统。 其中一个模型系统，美国牛蛙（ 牛蛙 ）的球囊，几十年来取得了一般化洞察听觉和前庭系统的功能。小囊是一个混合功能的器官，在低频听力和地震的感觉都感觉的作用。所述球囊的感觉细胞是其毛细胞，将机械能转换专门的换能器到我们的听觉和前庭器官内的电信号。从每个毛细胞的顶端表面伸出是一个机械敏感毛发束，其包括称为纤毛放大微绒毛的分级刷毛束。相邻的纤毛的尖端由丝状尖链路蛋白质互相连接，机械门离子通道响应于机械性刺激2，3。虽然听觉和前庭器官的不同类型的刺激做出反应，他们都有一个共同的检测机制。这种共性underlies通过球囊牛蛙研究获得的成毛细胞机械传导的许多见解。例如，毛细胞的活性过程已被广泛这个器官4，5，6，7的研究，并且发束采用了能量消耗的过程，以产生机械工作。它不仅被证明，毛细胞产生积极工作6，但在活动过程基本不同的机制和毛细胞的调谐特性已经通过牛蛙acousticolateralis器官的研究亮相。这些包括有源毛发束蠕动8和毛细胞电共振9，10，在球囊11和频率选择性在所述水陆两用车辆乳头毛细胞的色带突触12。 牛蛙的球囊吸引的原因很多感官神经科学家。不同于哺乳动物耳蜗，这个器官位于易于接近耳胶囊内。其次，本机关内毛细胞可以在适当的条件下，13保持了几个小时健康，14。这使得experimentat离子对这些细胞在相对其同行哺乳动物长的时间尺度。三，器官熊小弧度，允许容易操控。第四，每个器官包括一千或更多毛细胞15，同时提供高通量和定位一组适当的毛细胞对于给定实验的可能性高。最后，牛蛙的球囊很容易由于其毛细胞这个器官和大尺寸的薄可视化。 这些特性提供了感觉细胞的牛蛙的球囊内的研究具有通用性。取决于手边的问题，有几个实验制剂中的一个可从球囊中获得。其中最简单的是单腔室制备。在这里，球囊固定在充满人工外淋巴液，丰富的钠和高钙盐水室。这种准备使毛细胞电流和基本的发束力学研究。第二结构中，两个腔制剂，可用于研究自然发束运动。这里毛细胞的顶面暴露于钾富和钙差盐水称为人工内淋巴，而基底外侧面在人工外淋巴沐浴。这两个隔室模拟生理盐水， 在体内的装置和提供允许发束自发振荡的环境。 我们在本文中描述了牛蛙的球囊为它的感觉毛细胞生物物理研究的准备。我们首先提供了从青蛙的内耳这个器官的孤立的详细描述。然后，我们描述了两个单组分和双室的实验准备工作和包括每个配置代表性的结果。

<table><tbody><tr><td>&lt;strong&gt;Common to both preparations&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stereo-dissection microscope</td><td>Leica</td><td>MZ6</td><td>Other sources can be used</td></tr><tr><td>Tricaine methanesulfonate</td><td>Sigma</td><td>E10521</td><td>Other sources can be used</td></tr><tr><td>Metal pithing rod</td><td>Fine Science Tools</td><td>10140-01</td><td></td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>15000-03</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Glass Pasteur pipette and bulb (x2)</td><td>Fisher Scientific</td><td>22-042816</td><td></td></tr><tr><td>Fine eyelash mounted on a hypodermic needle</td><td>Fisher Scientific</td><td>22-557-172</td><td></td></tr><tr><td>Dow-corning vacuum grease</td><td>Fisher Scientific</td><td>14-635-5C</td><td></td></tr><tr><td>Syringe for vacuum grease</td><td>Fisher Scientific</td><td>14-829-45</td><td>Other sources can be used</td></tr><tr><td>35 mm Petri dish (x2 - 3)</td><td>Fisher Scientific</td><td>08-772A</td><td>Other sources can be used</td></tr><tr><td>Micropipette puller</td><td>Sutter</td><td>P-97 or P-2000</td><td></td></tr><tr><td>120 V Solenoid puller</td><td></td><td></td><td>Home-made, see parts list</td></tr><tr><td>Sputter coater</td><td>Anatech USA</td><td>Hummer 6.2</td><td></td></tr><tr><td>Current source for iontophoresis</td><td>Axon Instruments</td><td>AxoClamp 2B</td><td>Other sources can be used</td></tr><tr><td>Piezoelectric actuator</td><td>Piezosystem Jena</td><td>P-150-00</td><td></td></tr><tr><td>Amplifier for piezoelectric actuator</td><td>Piezosystem Jena</td><td>ENV800</td><td></td></tr><tr><td>Borosilicate glass capillary</td><td>World Precision Instruments</td><td>1B120F-3</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For one-chamber preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microelectrode amplifier</td><td>Axon Instruments</td><td>AxoClamp 2B</td><td>Can be used for iontophoresis and microelectrode recordings simultaneously</td></tr><tr><td>Magnetic pins (x2)</td><td></td><td></td><td>Home-made, see parts list</td></tr><tr><td>Open-top chamber with magnetic sheet</td><td></td><td></td><td>Home-made, see parts list</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For two-chamber preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Upper chamber</td><td></td><td></td><td>Supplementary file 1</td></tr><tr><td>Troughed lower chamber</td><td></td><td></td><td>Supplementary file 2</td></tr><tr><td>Aluminum foil</td><td>Fisher Scientific</td><td>01-213-100</td><td>Other sources can be used</td></tr><tr><td>Mounting block</td><td></td><td></td><td>Supplementary file 3</td></tr><tr><td>Wooden applicator sticks</td><td>Fisher Scientific</td><td>23-400-112</td><td>Other sources can be used</td></tr><tr><td>Teflon sheet</td><td>McMaster-Carr</td><td>8545K12</td><td>For teflon applicator</td></tr><tr><td>Cyanoacrylate glue</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Lab tissues (Kimwipes)</td><td>Fisher Scientific</td><td>06-666A</td><td>Other sources can be used</td></tr><tr><td>Gentamicin sulfate</td><td>Sigma-Aldrich</td><td>G1914</td><td>Other sources can be used</td></tr><tr><td>Quick-setting epoxy</td><td>McMaster-Carr</td><td>7605A18</td><td></td></tr><tr><td>18 mm glass coverslips</td><td>Fisher Scientific</td><td>12-546</td><td>Other sources can be used</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Saline components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-3</td><td>Other sources can be used</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P4504-500G</td><td>Other sources can be used</td></tr><tr><td>CaCl&lt;sub&gt;2 &lt;/sub&gt;&amp;bull; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>10035-04-8</td><td>Other sources can be used</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375-100G</td><td>Other sources can be used</td></tr><tr><td>D-(+)-glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td>Other sources can be used</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Parts lists for home-made equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Solenoid puller&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Solenoid</td><td>Guardian Electric</td><td>A420-065426-00</td><td>Other sources can be used</td></tr><tr><td>Foot-pedal switch</td><td>Linemaster</td><td>T-51-SC36</td><td>Other sources can be used</td></tr><tr><td>Pipette holder</td><td>World Precision Instruments</td><td>MEH900R</td><td>Other sources can be used</td></tr><tr><td>Coarse manipulator</td><td>Narishige Group</td><td>MM-3</td><td>Other sources can be used</td></tr><tr><td>Platinum wire</td><td>Alfa Aesar</td><td>25093</td><td>Other sources can be used</td></tr><tr><td>Power supply</td><td>Leica</td><td>Z050-261</td><td>Other sources can be used</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Magnetic pins&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Epoxy</td><td>McMaster-Carr</td><td>7556A33</td><td>Other sources can be used</td></tr><tr><td>1 mm thickness aluminum</td><td>McMaster-Carr</td><td>89015K45</td><td>Other sources can be used</td></tr><tr><td>Insect pins</td><td>Fine Science Tools</td><td>26000-40</td><td>Other sources can be used</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Open-top magnetic chamber&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Flexible magnetic strip</td><td>McMaster-Carr</td><td>5759K75</td><td>Other sources can be used</td></tr><tr><td>1 mm thickness aluminum</td><td>McMaster-Carr</td><td>89015K45</td><td>Other sources can be used</td></tr></tbody></table>

physiological preparation of hair cells from the sacculus of the american bullfrog (rana catesbeiana)

听力和平衡的研究停留在从模型系统的生物物理研究中得出的见解。一个这样的模型，美国牛蛙的球囊，已成为听觉和前庭研究的中流砥柱。有这种器官的研究揭示细胞如何感觉毛可以主动从环境中检测到的信号。因为这些研究中，我们现在更好地了解机械门控和毛细胞的转导通道，钙的机械适应角色的定位，和毛细胞电流的身份。这种高度开放的器官继续提供洞察毛细胞的运作。在这里，我们描述了它的毛细胞生物物理研究牛蛙的球囊的准备。我们包括完整的解剖程序，并为在具体情况下的球囊的准备具体的协议。我们还包括使用该制剂的代表性结果，其中的计算发束的瞬发力 - 位移关系，一个包的自激振荡的测量。

牛蛙的球囊的感觉上皮可以以各种配置可以采用探测毛细胞的生理学。因为组织是相对平坦的，它可以被安装在两一个和两个腔室的制剂。一室配置提供了毛细胞的电生理和微机械录音进行简单的设置。双室制备代替模拟两内淋巴和上分别毛细胞的顶和底侧面外淋巴室中。这些车厢一起为机械传导由毛细胞研究的生理相关的环境。 毛细胞的敏感性和转导特性背后其对机械刺激的电响应。探测这些功能，我们同时来自个体毛发记录细胞中的束的位置，并细胞的受体电位（ 图2）。我们首先连接的柔性玻璃纤维到一个发束的kinociliary灯泡施加力脉冲。然后，我们测量使用双光电二极管系统2（ 图2A）的发束的位移。我们通过同时用锋利的微电极刺穿细胞获得的毛细胞的潜力。我们通过绘制通过对发束的相应的位移（ 图2B）的每个机械刺激引起的峰值电压响应而得到的位移响应曲线。毛细胞的电响应饱和为正，负位移极值。用负位移步骤的膜电位的降低表明静止向内机械传导电流的存在。此静止电流由钙离子的作用而在两个快速和慢速自适应17调制， <sup系列="外部参照"&gt; 18，19，20，21。 毛细胞的行为不仅取决于其电气性能，而且还取决于它的感觉毛束的微观力学。双室结构模仿体内内淋巴和外淋巴的分离，为一个发束的力学的研究提供了理想的条件。在这些条件下，用除去耳石膜，头发束可摆动自发6。在这里，我们采用的双室制备，以评估单独的捆的微机械。我们通过铸造其影子的双光电二极管位移监控器（ 图3）上记录的一个发束的自发振荡。为了评估机械传导氨基糖苷类抗生素庆大霉素的作用，我们离子电渗RELE<html>直接ASED庆大霉素到发束（ 图3）。释放庆大霉素的浓度与通过微量传递电流成比例地上升。庆大霉素抑制发束的振荡并导致静态朝其高大的侧束的偏移量。这些效应反映庆大霉素的作用，维持机械传导通道的开放状态，同时阻止其渗透孔的开放通道阻滞剂。带电的化学品的离子电渗疗法允许局部和以各种浓度在没有流体流动引起的机械破坏的化学物质的量化释放并且因此非常适合于机械敏感细胞器的研究，如在发束22上 。 发束的自发运动源于适应和非线性束刚度7之间的相互作用，外部参照"&gt; 8，23，24，这自发运动是一个发束的活动过程，该信号能量转换成机械功，克服粘性阻力的签名。发束已经显示呈现在前庭2，听觉非线性瞬时刚度25，以及侧线系统26。 我们直接从牛蛙的球囊（ 图4）测得的个别毛发束的瞬时刚度。为了实现这一点，我们耦合的柔性玻璃纤维的发束的kinociliary灯泡（ 图4A）的尖端。我们通过置换纤维的基本交付部队发束。通过刺激纤维施加到发束上的力对应于纤维的浅的位移之间的差e和技巧，再乘以纤维的硬度2，27。在一系列的力的传送脉冲揭示施加在捆和包的随后的位移的力之间的关系。这个力-位移关系的斜率对应于发束的瞬时刚度（ 图4A）。 该方法使我们能够测量单个包的瞬时刚度作为其偏转（ 图4B）的功能。瞬时力位移曲线显示一个非线性关系，揭示了束的非线性刚度的范围内围绕其静止位置约20nm。在此范围之外，发束的行为类似于虎克材料，其硬度为大幅度偏转线性。 这些结果demonst率牛蛙球囊的多功能性毛细胞生理学研究。使用这些和其它制剂，可以在信息朝向脑束的发送探索在多个阶段机械传导。 <img alt="图1" src="/files/ftp_upload/55380/55380fig1.jpg" /> 图 1: 牛蛙的内耳解剖。 （ 一 ）从腹侧查看牛蛙的上腭允许耳咽管（圈子）的标识。覆盖上腭的右侧的皮肤侧向反射揭示内耳（虚线框）的位置。对青蛙的颞骨的腹侧软骨（B）的除去将打开听囊（虚线）。 （C）显示的是听囊的更高放大倍数图像，其中，所述球囊，lagena，CN第八和SACC<html> ular神经可以容易地识别。 （D）除去内耳器官后听囊的视图揭示了半规管的位置。 （E）拆除隔离内耳的球囊，lagena，第八和第脑神经（CN VIII）后，可以很容易地识别。 （ 六 ）独立的小囊具有囊状神经和耳石膜躺在上面的感觉上皮短树桩。标签对应于第（i）小囊，（ⅱ）lagena，（ⅲ）CN VIII，（ⅳ）囊状神经，以及（v）耳石膜。轴标签P和A分别对应于后部和前方向。刻度条表示1厘米（A，B），1-毫米（C，D，E）和400微米（F）。 <a href="http://ecsource.jove.com/files/ftp_upload/55380/55380fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 的"目标="_空白"&gt;请点击这里下载这个数字的向量版本。 <img alt="图2" src="/files/ftp_upload/55380/55380fig2.jpg" /> 图2: 位移响应曲线单毛细胞。 （A）一种玻璃刺激纤维的尖端耦合到发束的kinociliary灯泡和纤维的基九个离散的步骤，随后位移。束的位置被跟踪的双光电二极管的系统上，使用微电极，其输出通过放大器传递在桥模式及其受体电位同时测量。电极的尖端电阻为95MΩ和包的静息膜电位为-47 mV的。 （ 二 ）包的受体电位作为其位移函数的曲线揭示了包的响应及其之间的非线性关系位置。每个点对应于平均电势和平均位移超过2.5毫秒的时间窗口中，机械刺激的发作后开始2.5毫秒。每种颜色代表一组对应于同排量的脉冲时间序列。 <a href="http://ecsource.jove.com/files/ftp_upload/55380/55380fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <a href="http://ecsource.jove.com/files/ftp_upload/55380/Figure2_Xd_Xc_Vm_version4.eps" target="_blank">请点击此处下载这个数字的向量版本。</a> <img alt="图3" src="/files/ftp_upload/55380/55380fig3.jpg" /> 图3:庆大霉素对自发发束振荡的影响。一个发束中的两腔室制备的自发运动，使用了双光电二极管系统记录。在没有庆大霉素离子电渗疗法释放的（0 NA），头发束显示对称振荡。作为电流值通过填充用500mM硫酸庆大霉素生长（10 nA的，20 NA），毛束偏移落在以剂量依赖性方式与束朝向其高大边缘偏移更长的频率的电离子透入吸液管通过的时间段。 <a href="http://ecsource.jove.com/files/ftp_upload/55380/55380fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <a href="http://ecsource.jove.com/files/ftp_upload/55380/Figure3_GentaSpontOsc_version2.eps" target="_blank">请点击此处下载这个数字的向量版本。</a> <img alt="图4" src="/files/ftp_upload/55380/55380fig4.jpg" /> 图4. 计算一个发束的瞬时 刚度。刚度ķF（A）刺激纤维（红色）连接Ť澳kinociliary灯泡（棕色）个人发束（黄色）的。位移光纤的基部已知距离X F使得移动距离X B中的包。纤维和纤维束的位移之间的差异成比例的力施加在捆通过刺激纤维，F F。在一系列的力的重复该产生的瞬时力 - 位移关系（右），其斜率的对应于发束的瞬时刚度。 （B）的一个单独的束进行脉冲发生，测定（蓝点）后，以迫使第一50毫秒内增加幅度和其位移的脉冲。这里发束显示的范围内的围绕其搁置位置大约20nm的非线性瞬时刚度。红色曲线对应于一个适合的关系:F =ķ* X - 60 * Z *（1 /（1 + EXP（ - Z *<html> （X - X 0）/（K B * T）））+ F 0，其中F是施加在束上的力，X是包的位移中，k = 790±51μN∙M -1是该包的恒定刚度所有通道时，无论是封闭或开放，Z = 0.43±0.04 PN是一款单门弹簧力，X 0 = 2±1.9纳米的bundle的位置，在该公司渠道的50％是开放的，K B是玻耳兹曼常数，T是温度，和F 0 = 11.7±1.3 PN是偏移力。拟合具有确定的0.98的系数。刺激纤维具有107μN的刚度∙M-1。 <a href="http://ecsource.jove.com/files/ftp_upload/55380/55380fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ES / ftp_upload / 55380 / Figure4_v5.eps"目标="_空白"&gt;请点击这里下载这个数字的向量版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> 溶质 </td><td rowspan="2"> 式重量（g / mol）的 </td><td colspan="2"> 添加到1升 </td></tr><tr><td> 人工外淋巴液 </td><td> 人工淋巴 </td></tr><tr><td>氯化钠</td><td> 58.4 </td><td> 6.54克</td><td> 0.117克</td></tr><tr><td>氯化钾</td><td> 0.149 </td><td> 0.149克</td><td> 8.62克</td></tr><tr><td> 氯化钙 ⦁2H 2 O </td><td> 147 </td><td> 2毫升1M的氯化钙2库存</td><td> 1M的氯化钙2股250微升</td></tr><tr><td> HEPES </td><td> 238.3 </td><td> 1.19克</td><td> 1.19克</td></tr><tr><td> D - （+） - 葡萄糖</td><td> 180.2 </td><td> 0.541克</td><td> 0541克</td></tr></tbody></table> 表1.解决方案解剖和实验准备。显示在此表是用于人工外淋巴和在解剖和在一个或两个腔室制剂中使用人造淋巴溶液的配方。该解决方案应该将pH值调至7.2 - 7.4与约2毫升氢氧化钠（外淋巴）或2毫升KOH（淋巴）的。该渗透强度应为约230毫摩尔·kg -1时，由于不完整的离子离解。

Watch this Scientific Journal Video about Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana at JoVE.com

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

美国牛蛙的（ 牛蛙 ）允许小囊毛细胞生理学的直接检查。在这里，解剖和准备牛蛙的球囊对生物物理研究的描述。我们显示从这些毛细胞代表的实验中，其中包括一个包的力 - 位移关系的计算和测量它的非受迫运动。

从美国牛蛙的小囊（毛细胞的生理准备<em&gt;牛蛙</em&gt;）

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

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Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

16.7K 次观看.  The Rockefeller University. 美国牛蛙的（ 牛蛙 ）允许小囊毛细胞生理学的直接检查。在这里，解剖和准备牛蛙的球囊对生物物理研究的描述。我们显示从这些毛细胞代表的实验中，其中包括一个包的力 - 位移关系的计算和测量它的非受迫运动。 

视频: Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

与耳蜗内毛细胞传入树突：多泡发布监测功能区突触的突触后录音

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

科研

神经科学

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 ...

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  ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

发育生物学

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1&#8211;2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

活体和功能性鼠耳蜗毛细胞中的德特贴标签和吸收

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET ...

生物工程

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair cells.

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia ...

从美国牛蛙的小囊（毛细胞的生理准备

摘要

探索更多视频

此视频中的章节

与耳蜗内毛细胞传入树突：多泡发布监测功能区突触的突触后录音

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

膜片钳记录在内耳毛细胞的分离斑马鱼

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

在蛋和卵外研究中研究鸟类内耳发育的方法

活体和功能性鼠耳蜗毛细胞中的德特贴标签和吸收

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

幼虫斑马鱼外系头发细胞的活体钙成像

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

从美国牛蛙的小囊（毛细胞的生理准备

摘要

探索更多视频

此视频中的章节

与耳蜗内毛细胞传入树突：多泡发布监测功能区突触的突触后录音

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

膜片钳记录在内耳毛细胞的分离斑马鱼

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

活体和功能性鼠耳蜗毛细胞中的德特贴标签和吸收

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

幼虫斑马鱼外系头发细胞的活体钙成像

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

在蛋和卵外研究中研究鸟类内耳发育的方法