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毫升的甘油的人工脑脊液（ACSF）组成的（以mM计）：26的NaHCO 3，11葡萄糖，250份甘油，2.5氯化钾，1.2的NaH 2 PO 4，1.2的MgCl 2，CaCl 2的 2.5。在此之前加入的CaCl 2，气体卡波金（95％O 2和5％CO 2）的解决方案，以建立应用7.4和H的，避免钙质沉淀（混浊）。冻结的溶液在-80℃的冰箱中45分钟，使冰形成浆料。 </li><li>深麻醉小鼠通过腹腔注射氯胺酮（100毫克/千克）（帕内尔，亚历克斯，澳大利亚）。 </li><li>一旦后肢捏反射消失杀头鼠标使用的是锋利的不锈钢剪刀，并用刀片（四舍五入＃22），暴露颅骨矢状切开皮肤。在这一点上和整个步骤2.3-2.8颅，脑，及相关的前庭器官应保持尽可能凉爽，由冰冷学联组织在普通的应用程序。 </li><li>使用标准模式剪刀尖臂（FST），加拿大北温哥华做一个小切口在颅骨的Lambda和切沿矢状缝。确保大脑不是“拖”在此步骤中的剪切刀片。 </li><li>小心剥离的顶骨，横向和枕骨posterio的的RLY使用，浅弯皮尔逊咬骨钳（FST）。 </li><li>然后，使用一个小的不锈钢刮勺轻轻抬起大脑从中间和后颅窝的表面，暴露vestibulocochlea的神经（CNVIII）内耳和脑干中的一对细的虹膜剪切的中间。切割神经初级传入和他们与毛细胞的轴突最大限度地减少不必要的紧张。 </li><li>第八CN横断脑全盘被删除。 </li><li>前庭迷路中颅窝清晰可见，与耳蜗指向前内侧。咬骨钳才轻轻抓住前半规管和横向拉切除两侧的前庭迷路。 </li><li>沉浸在解剖盘冰冷的，连续毒气学联第2.1节中描述的的切除迷宫（ 图1）。在立体显微镜下，按住迷宫烹调的基础上，通过抓住耳蜗。用细镊子刮去骨覆盖前半规管壶腹。一旦实现一个小洞，在骨开始滑动骨离壶腹。时应注意，不要推，通过骨钳，因为这可能会导致损坏底层膜迷路和感觉上皮。的前部和相邻的水平半圆形膜管壶腹继续 ​​执行此过程，直到露出（ 图2）。 </li></ol><img alt="图1" src="/files/ftp_upload/50471/50471fig1.jpg" /> 图1。小鼠离体前庭迷路。左面板）示意图小鼠离体前庭迷路。重要的参考点访问前庭感觉上皮细胞，耳蜗，前壁，水平半规管实验室ELED。星号表示半规管壶腹的前庭感觉上皮。右面板）的显微照片，从1个月大的小鼠的孤立前庭迷路。 <img alt="图2" src="/files/ftp_upload/50471/50471fig2.jpg" /> 图2。曝光的膜前庭迷路。骨覆前水平半规管壶腹被划伤，露出黑色/ ​​棕色斑点膜壶腹及相关壶腹的神经（CNVIII）。底部面板中的示意性表示的结构中的高亮区域的显微照片，显示的关系半规管的管道壶腹和CNVIII的。 <ol start="10"><li>用细镊子，轻轻抬起壶腹及相关囊远离骨迷路，确保中部地区壶腹（CONTA矿业感觉上皮）没有被损坏。在某些情况下，可能需要削减的半圆形的膜管与虹膜剪，以释放从骨壶腹近端部分。 </li><li>包含两个壶腹和囊Leibovitz的L-15培养基（Sigma-Aldrich公司，圣路易斯，密苏里州）到培养皿充满了黑社会转移。使用光纤“背光”组织。这使得感觉上皮内壶腹嵴清晰的可视化。 </li><li>斑点的椭圆囊的“屋脊”之称的罚款虹膜剪小心地做一个切口。屋顶的前，后水平壶腹继续通过此切口。使切口尽可能靠近边缘的感觉上皮尽可能不接触（ 图3A）。确保有膜覆盖的感觉上皮无件（ 图3B） </li><li>孤立半完整的准备转移到一个小GLASL-15的媒体充满了S-有底录音室。称取下来使用网格固定的扁平的U形的铂丝（ 图3B）的细尼龙纤维编制。理想情况下，纤维的网格不应该覆盖在感觉上皮，但是在某些情况下，更多的稳定性是必需的，横切的感觉上皮可能优选的单纤维。 </li></ol><img alt="图3" src="/files/ftp_upload/50471/50471fig3.jpg" /> 图3。前庭感觉上皮。示意图的半完整的准备和电极配置的分离的半完整的制备 。壶腹嵴上覆有“封顶”的感觉上皮（绿色）的表面露出半完整的&#39;黑社会&#39;准备前（AC）和水平（HC）嵴（囊的显微照片。掩盖是后腿交流）。注意用于录音室的基础上制备固定的尼龙纤维。比例尺：100微米。C.记录电极放置在一个单独的前庭毛细胞。比例尺：15微米。 3。电<ol><li>灌注的半完整的连续含氧的L-15培养基的制备。该媒体包含pH指示剂（酚红）和颜色的介质应监测整个录音。与氧的pH值应为7.3-7.4，对应的红色的颜色。 </li><li>从1.5毫米（1.19 mm内径）硼硅酸盐玻璃上的微量拉马（成茂，日本，PP-830型），实现了使用一个两步骤的协议（加热步骤1：72;和热步骤2：50）准备记录移液管最终3-4MΩ阻抗。 </li><li>填充有3-4毫米的氟化钾为基础的内部含有（以mM）110 KF，氯化钾12 27氢氧化钾，氯化钠，10 HEPES，10 EGTA，1.8 MgCl 2的溶液的吸液管柄，3个D-葡萄糖，钠-ATP和2，pH为7.4，用KOH。 </li><li>包裹柄部的移液管的2-3倍，并尽可能远的前端尽可能用封口膜的细带绝缘的电极，并缩小移液器电容。 </li><li>吸管放置在感觉上皮一个堂堂正正的显微镜（奥林巴斯BX51）在低功率放大倍率（5倍）。切换到高功率（40X）和一个附加的CCD相机，可视个别前庭毛细胞。 </li><li>仓移液管上的膜用显微操纵器（萨特仪器，加利福尼亚州，美国）的可视化毛细胞（ 图3C）。一旦千兆欧姆密封件来实现，适用于通过吸液管保持器上的吸入口的负压用少量的细胞膜破裂。 </li><li>请使用标准的8-9技术的全细胞电压钳记录。 </li></ol> 4。双光子显微镜<ol><li>准备一个0.9％的生理盐水溶液含有5毫米俄勒冈绿488 BAPTA-1（OGB-1; hexapotassium盐; Invitrogen公司，德国）。 </li><li>虽然仍然淹没，放置到一小块滤纸（4X4毫米，厚0.8微米的微孔，德国），确保，感觉上皮通畅的从上面的“封顶”半完整的黑社会准备。 </li><li>再有5微升5mM的溶液中合成的硅酸钙转移到盐水覆盖基铂电极（7微升）的脉冲发生器和一个宽带放大器的组合构成的电穿孔，滤纸及其制备和盖指示剂染料俄勒冈绿488 BAPTA-1（ 图4）。 </li></ol><img alt="图4" src="/files/ftp_upload/50471/50471fig4.jpg" /> 图4。散装电。电配置的横截面示意图。半完整的制剂（*）被放置在一张滤纸浸入DY钙E（俄勒冈绿488 BAPTA-1）和2铂电极之间施加电流。改编自Briggman和欧拉，2011年10。 <ol start="4"><li>在距离约2毫米的基极，带来顶级的铂电极平行小心，不要破坏的准备方向与俄勒冈绿488 BAPTA-1解决方案时。 </li><li>整个编制参数的电流，+13 V，10毫秒的脉冲宽度，频率1赫兹，10个方波脉冲电流脉冲通过一个简短的10-11。 </li><li>在底部的玻璃有底录音室充满了L-15培养基，放置一个小的硅油和位置的半完整的制剂轻拍到它，从而也确保了感觉上皮来自上述通畅。为了确保整个成像稳定性，在准备取代尼龙网格如上所述（见2.13点）。 </li><li>使用双光子显微镜，光学录音自发neous Ca2 +活动的感觉上皮（ 图7），使用标准的成像协议10-11。 </li></ol>

<ol>
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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>

作者宣称，他们有没有竞争经济利益。

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 ...

an-isolated-semi-intact-preparation-mouse-vestibular-sensory

Research

JoVE Journal

Neuroscience

11.8K 次观看.  University of Sydney. 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。

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

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

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 ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

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 ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

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

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

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

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

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 ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

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 methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

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 ...