Name: an isolated semi-intact preparation of the mouse vestibular sensory epithelium for electrophysiology and high-resolution two-photon microscopy
Uploaded: 2013-06-13T12:00:00
Description: Watch this Scientific Journal Video about An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy at JoVE.com

这项工作的资金提供了一个加内特凋谢和罗德尼·威廉姆斯纪念基金会项目资助R. Lim和AJ营。

1。动物<ol><li>小鼠获得了来自澳大利亚啮齿动物中心（ARC澳大利亚珀斯）和悉尼大学博世动物设施在一个正常的12小时的光/暗周期与环境富集举行。所有的实验是由悉尼大学动物伦理委员会的批准。 </li><li>雄性和雌性小鼠（C57/BL6）被用于所有的实验中是常用的，因为该菌株进行遗传操作的背景，可以视为等同野生型 8-9。 </li></ol> 2。组织编制<ol><li>准备300毫升...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+glycine+receptor+function+reduces+excitability+of+mouse+medial+vestibular+nucleus+neurons.">Attenuated glycine receptor function reduces excitability of mouse medial vestibular nucleus neurons.</a> Neuroscience. 170, 348-360 (2010).</li><li>Briggman, K. L., Euler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+electroporation+and+population+calcium+imaging+in+the+adult+mammalian+retina.">Bulk electroporation and population calcium imaging in the adult mammalian retina.</a> J. Neurophysiol. 105, 2601-2609 (2011).</li><li>Briggman, K. L., Helmstaedter, M., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wiring+specificity+in+the+direction-selectivity+circuit+of+the+retina.">Wiring specificity in the direction-selectivity circuit of the retina.</a> Nature. 471, 183-188 (2011).</li><li>Rennie, K. J., Streeter, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+currents+in+isolated+vestibular+afferent+calyx+terminals.">Voltage-dependent currents in isolated vestibular afferent calyx terminals.</a> J. Neurophysiol. 95, 26-32 (2006).</li><li>Hudspeth, A. J., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+voltage-+and+ion-dependent+conductances+in+saccular+hair+cells+of+the+bull-frog,+Rana+catesbeiana.">Kinetic analysis of voltage- and ion-dependent conductances in saccular hair cells of the bull-frog, Rana catesbeiana.</a> J. Physiol. 400, 237-274 (1988).</li><li>Rennie, K. J., Ashmore, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+currents+in+isolated+vestibular+hair+cells+from+the+guinea-pig+crista+ampullaris.">Ionic currents in isolated vestibular hair cells from the guinea-pig crista ampullaris.</a> Hear. Res. 51, 279-291 (1991).</li></ol>

我们平衡感的机制已经收到了有限的关注相比，与其他的感官系统， 例如视觉和听觉系统。的调查研究，在前庭平衡功能的变化，大多数都集中行为的措施，包括前庭眼反射，平衡的前庭毛细胞本身的基本构建块不完整的知识。这些研究都集中在毛细胞几乎每年都这样做急性分离的毛细胞，从他​​们的家乡环境中删除。在这篇文章中，我们展示了一个半完整编制的鼠标前庭感觉上皮细胞...

尽管前庭系统的重大贡献，我们的日常生活中，一个清醒的认识过程负责前庭功能明显下降，随着年龄的增长仍然是难以捉摸的。这种缺乏知识的原因之一是，在前庭功能下降，几乎完全被探索使用行为分析，包括前庭眼反射（VOR），精确的外在前庭功能的指标，但提供有限的内在因素的变化洞察。这是我们了解前庭毛细胞功能健康，疾病或衰老的一个主要障碍。 虽然已经有许多研究个别前庭毛细胞，一大缺憾一直依赖急性毛细胞制剂，其中毛细胞和从他们的正常的环境中，通过机械和/或酶处理甚至萼传入终端被删除。这种方法不可避免地BLY破坏毛细胞和花萼，毛细胞和支持细胞之间的微妙微架构。半完整的准备1-5和鼠标一个孤立的迷宫准备6随着时代的发展，现在有机会学习各种形式的突触通信的条件下，更接近于体内 。事实上，林等人 （2011）表现出显着的差异，在我前庭毛细胞相比那些仍然嵌入神经 ​​上皮的全细胞电流记录急性分离型。具体而言，钾被认为积聚在细胞间隙，毛细胞和萼传入神经 ​​之间，并显着改变毛细胞响应7。这种类型的信息将是不可能取得未经半完好无损的在前庭感觉上皮这里描述的制备。我们证明了半完整编制鼠标嵴3，显示全细胞膜片电，代表结果和双光子钙成像。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Leibovitz medium L-15</td>
 <td>Sigma Aldrich</td>
 <td>L4386-10X1L</td>
 <td></td>
 </tr>
 <tr>
 <td>BAPTA-1-oregon green</td>
 <td>Invitrogen</td>
 <td>O6806 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT </td>
 </tr>
 <tr>
 <td>Stereo microscope</td>
 <td>Leica Microsystems</td>
 <td>A60S</td>
 <td></td>
 </tr>
 <tr>
 <td>Upright microscope</td>
 <td>Olympus</td>
 <td>BX51WI</td>
 <td></td>
 </tr>
 <tr>
 <td>Two-photon microscope</td>
 <td>Olympus/La Vision</td>
 <td>BX51WI/ TriMScope II</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont #5 SF Forceps</td>
 <td>FST</td>
 <td>11252-00</td>
 <td></td>
 </tr>
 <tr>
 <td>Friedman-Pearson Rongeurs</td>
 <td>FST</td>
 <td>16221-14</td>
 <td></td>
 </tr>
 <tr>
 <td>Standard Pattern Scissors</td>
 <td>FST</td>
 <td>14001-12</td>
 <td></td>
 </tr>
 <tr>
 <td>InstraTECH A-D converter</td>
 <td>HEKA</td>
 <td>ITC-18</td>
 <td></td>
 </tr>
 <tr>
 <td>Sutter Micromanipulator</td>
 <td>Sutter</td>
 <td>MP-225/M</td>
 <td></td>
 </tr>
 <tr>
 <td>multiclamp amplifier</td>
 <td>Axon Instruments</td>
 <td>700B</td>
 <td></td>
 </tr>
 <tr>
 <td>Data acquisition software (electrophysiology)</td>
 <td>Axograph</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Imspector Data acquisition software (two-photon)</td>
 <td>Max Planck innovation</td>
 <td>N/A</td>
 <td></td>
 </tr>
 </tbody>
</table>

an isolated semi-intact preparation of the mouse vestibular sensory epithelium for electrophysiology and high-resolution two-photon microscopy

了解前庭毛细胞的功能在正常条件下，或外伤，疾病，老化破坏这种功能如何预防方法和/或新的治疗策略的发展是一个重要的步骤。然而，大多数看着前庭功能异常的研究还没有在细胞水平上，但主要集中在行为检测前庭功能障碍，如步态分析和前庭眼反射性能。虽然这项工作已经取得了宝贵的数据，会发生什么事情出错时，几乎没有信息搜集功能障碍的根本原因。集中在细胞和亚细胞过程背后前庭功能的研究，大部分都依赖于毛细胞急性分离的，没有他们的突触连接和支持细胞环境。因此，重大的技术挑战已经在准备访问极其敏感的前庭毛细胞aration至少打乱，生理。在这里，我们展示了一个半完整编制的鼠标前庭感觉上皮细胞，保留局部微环境，包括毛细胞/初级传入神经复合物。

前庭毛细胞的电生理特性依赖于它们所嵌入的7上的复杂的微体系结构。 图5示出了半完整的前庭上皮细胞的制备，可用于区分不同的我发细胞（ 图5A），II型毛细胞（ 图5B），萼初级传入神经 ​​（ 图5C），基于特征的全细胞电导。这些特性包括去极化过程中一个明显的“凹陷”和大型“崩溃”的尾电流I型毛细胞，存在暂时...

Watch this Scientific Journal Video about An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy at JoVE.com

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

前庭毛细胞功能分析是复杂的，最难的部分颅骨，颞骨岩部内位置较深。功能最全的毛细胞的研究已经用毛细胞急性分离。在这里，我们描述了一个半完整的小鼠前庭上皮细胞的电生理和双光子显微镜研究编制。

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 ...

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

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

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Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...