This work was supported by a Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (T.I.) and Grants-in-Aid for Scientific Research (C) from MEXT (S.T. and K.N).

该协议和使用实验动物被批准的机构动物护理和使用委员会在山梨和早稻田大学的大学。按照机构准则进行动物护理。 1. CPEC准备<ol><li>准备下列设备和材料:立体显微镜，最好是能够发送从底部照明;一对钟表匠钳（杜蒙＃3＃或4），火焰消毒，操作直剪，火焰消毒;疏导10厘米塑料盘含有20ml冰冷的Leibovitz L-15培养基; 35毫米玻璃底菜含2ml RT莱博维茨的L-15介质;的100毫升烧杯中含有70％的乙醇;新生小鼠的幼崽。 </li><li>简要地浸入一个新生小鼠在70％乙醇，并使用所述操作剪刀断头很快安乐死。 </li><li>在冰冷的Leibovitz L-15培养基，立即将头在无菌10厘米二SH。 </li><li>除去从使用一对钟表制造商镊子的颅盖的皮肤，剖开颅骨以暴露脑，然后切颅神经以分离全脑。 </li><li>脑转移到含有冰冷的Leibovitz L-15培养基的新菜和立体显微镜下观察，确保大脑完全浸入在介质中。 </li><li>设置大脑朝上背侧，并定向使大脑嗅球驻留在三点钟位置（右旋人）。与在左手镊子轻轻握住大脑。 </li><li>使用细镊子剥离（杜蒙＃3＃或4）在右手，切断胼胝体和实质下方连接大脑半球，沿大脑纵裂。 </li><li>轻轻大脑半球推到侧面和揭露横向脑裂。 </li><li>按捏出来个分离半球半球和丘脑两地电子实质。 </li><li>轻轻拉出附加到海马的侧面通过椎板affixa的侧脑室的脉络丛。 </li><li>转移孤立脉络丛含有新鲜莱博维茨L-15中型35毫米的玻璃底菜，并覆盖重量（材料清单）轻轻地握住组织到位。 </li></ol> 2.现场CPEC纤毛成像<ol><li>确认正确的紫外线（UV）和推断（IR）截止滤光器（S）以阻挡光线于400nm和大于700纳米，且该中性密度（ND）滤波器（25％和6％）短插入到光路倒置显微镜。 </li><li>调整物镜的焦点大致由眼睛，然后调整冷凝器，使得中心和聚焦，以符合科勒照明。插入一个适当的微分干涉对比（DIC）棱镜，DIC元件，以及在立分析器和偏振元件向右路径以符合DIC的光学。 </li><li>调整的视图由DIC棱镜位置的对比度，以使组织表面的结构是最容易识别的。如果目标小区的所有纤毛是能动的，不能由眼睛，因为它们的运动获得的运动纤毛的清晰视图。 </li><li>改变光路的摄像头，并删除ND滤镜增加了光功率。 </li><li>使用相机对焦模式调整的观点和重点领域。在重点，其中视频图像显示在监视器上，在实时性，运动纤毛的清晰视图不可用。 </li><li>使用相机以200Hz为0.1毫秒的所需时间（数秒至数分钟）的曝光时间。采集图像堆栈后，单框会显示清晰的纤毛结构。如果纤毛边缘模糊，提高帧速率或使用更短的曝光时间。 </li><li>在安乐死后，莱博维茨在25-60分钟记录CPEC纤毛的运动L-15培养基。 </li></ol>睫状运动的三分析<ol><li>手动跟踪每一个跳动的纤毛模式的电脑显示器上。在每一帧使用鼠标指针，这是组装每个纤毛的轨迹信息标记纤毛尖部位置。无论分析使用相同的软件或导出到用于进一步分析其他更普遍应用的轨迹信息。该分析步骤的效率在讨论中描述。 </li><li>分类轨迹为两种模式的运动，背面的往​​复或旋转，通过眼睛。 </li><li>使用下列公式计算睫状跳动频率（CBF）:[CBF =（每秒帧的数目）/（帧为单次搏动的平均数目）] 14，它可以从一个纤毛尖端运动图得到（ 图3 ）。重复此计算为多个纤毛打浆周期，因为在同一单元中的其他纤毛可与每个ciliu的运动干涉米，从而导致不​​均匀性。 </li><li>分析睫状单个细胞内跳动轴的角均匀性，定义为每条轨迹（ 图4）的打浆角度θ。对背的往复轨迹，适合纤毛尖的位置，以直线，并定义θ为线使得与x轴的角度。对于旋转轨迹，适合的位置，以椭圆形，并定义θ为椭圆的长轴使得与x轴的角度。配件的细节在代表结果部分所述。 </li><li>对于每一个轨迹的定量描述，计算广义长宽比AR。简言之，旋转轨迹由- θ和定义的AR作为沿x中的分布的宽度之间的比例-和y -axes（ 图4B）。细节显示在代表结果部分，和解释意义，并且参数的限制将在讨论中描述。 </li></ol> 4.样品制备SEM 注:SEM是评价以全面的方式纤毛上CPECs地位的重要手段。为了制作标本的SEM，一个标准程序之前报道15，使用了轻微的修改。 <ol><li>之前，从脑的解剖组织，在准备用聚乙烯帽5毫升的玻璃小瓶的固定液。固定剂由2％多聚甲醛，2.5％戊二醛（半Karnovsky氏溶液16）的0.1M磷酸盐缓冲液，pH 7.4中的。 </li><li>解剖出从脑组织如步骤1所述。 </li><li>简要地冲洗以在一个新的培养皿Hank氏平衡盐溶液（HBSS）中分离的组织，然后固定在玻璃小瓶中的固定剂的组织进行1小时，在室温下进行。使用一次性移液管和处理标本GEntly。漂洗在HBSS之后，将组织变得粘稠。 <ol><li>到试样转移到固定剂溶液，慢慢排出少量含有从移液管的组织作为液滴溶液，并加入到固定剂。 </li></ol></li><li>固定后，弃去固定剂和冲洗用磷酸盐缓冲液三次组织。 </li><li>浸入所述组织在10％蔗糖溶液洗出剩余的醛。以确保完全消除醛类，浸入样品溶液中的10分钟，然后用新鲜的10％蔗糖重复两次。这个步骤是重要的，以实现正确的后固定在随后的步骤。 </li><li>浸入所述组织中的1％四氧化锇在磷酸盐缓冲液进行30分钟的溶液中，然后放置在冰上后固定。判断osmification由样本的颜色的程度:当醛完全除去，所述样品是黑色的。 </li><li>双distille广泛洗净后固定的组织样本ð水数次。 </li><li>脱水通过浸入样品中的乙醇梯度浓度，通常为65％，75％，85％，95％，99％和100％，每10分钟。通过将分子筛到99.5％的乙醇从一个新购买的瓶子得到无水乙醇。用无水乙醇三次重复脱水。 </li><li>将脱水的样品成异戊酯，取代试剂为临界点干燥，10分钟。重复此步骤两次。该试剂迅速蒸发和样品会变得干燥，导致破坏表面张力。因此，不完全干燥的样品。 </li><li>乙酸异戊酯的最后交换后，除去大部分溶剂后，立即用铝箔包装的开口的玻璃小瓶中，并放置在干冰上的小瓶中。使用针或细镊子，使几个孔在箔覆盖小瓶的口，以使液态二氧化碳容易流动到在临界点干燥器小瓶。继续进行下一步骤尽快。 </li><li>在此步骤中，最小化的乙酸异戊酯的夹带进入干燥器的腔室，但不要让样品完全干燥临界点干燥前。此外，不要离开干冰的样本不必要长的时间，以避免霜冻对小瓶的形成。 </li><li>传输含有组织样品箔包裹的玻璃小瓶进入临界点干燥器，确保组织的表面结构保持完好，同时除去水中所含的组织。详细信息上操作的临界点干燥器可从制造商的说明来获得。 </li><li>仔细用牙签，以尽量减少机械损伤处理样品。所得干燥的组织样品是易碎。安装在使用离子溅射金属存根和外套钯金的样品。 </li></ol> 5.观察SEM <ol><li>通过SEM观察，并用数码相机记录影像配到扫描电子显微镜。 </li><li>数字图像数据传输到PC进行分析。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。本文旨在报告的详细方法，观察纤毛的蠕动孤立脉络丛组织。科学新奇已经报道在以往的研究1,8。

这种方法的观点虽然此处所描述的技术并没有提供纤毛比以前公开的方法的更详细的分析，该技术的意义驻留在系统和成本效益，可以很容易地应用于筛选任何类型的纤毛运动离体的简单性。特别是，TI Workbench提供了一个简单和友好的用户界面，使研究人员观察和更容易地分析纤毛蠕动。有效的自动跟踪方法还没有被开发用于低对比度的物体，如在视频增强对比度的DIC...

Cilia are hair-like projections on the surface of most vertebrate cells, which have attracted attention by medical researchers because of a class of diseases termed ciliopathies2&#8211;4. Despite the ubiquitous expression of the organelle, a wide variety of ciliary functions have been reported, including motility and biosensing. For example, motile cilia on the mucoepithelial surface transport mucus5 and epithelial debris to the outlet of tracts, thereby preventing disease by clearing the surface of epithelia. Moreover, during early developmental periods and embryonic stages, cilia regulate the proliferation of stem cells6, and are involved in the determination of left&#8211;right asymmetry of the vertebrate body7. 
Choroid plexus epithelial cells (CPECs) are derivatives of neuroepithelial cells that cover the surface of the choroid plexus tissue in the brain, which play important roles in maintaining homeostasis of the intracranial environment by production of cerebrospinal fluid (CSF). It has been previously demonstrated that CPECs have multiple non-motile cilia that regulate the production of CSF through G-protein-coupled receptors that are specifically concentrated on the cilia8. Although these cilia had been regarded as quiescent non-motile cilia, it was discovered that some CPEC cilia exhibit transient motility during the neonatal period1. This finding was quite important because it revealed that so-called non-motile cilia are not necessarily immotile from the beginning of development and might display transient motility during specific time windows, possibly in response to specific physiological demands and functions9. To precisely describe the motile nature of CPEC cilia, it is necessary to develop an ex vivo observation system that encompasses analysis of the kinetic profiles unique to CPEC cilia. 
With respect to motility, although several technical reports have described observations of the motile cilia of the tracheal epithelium5,10, motile single-cell flagella11, so-called conventional motile cilia12, and nodal cilia13, detailed analytical methods applicable to relatively undulated structures such as the choroid plexus have not been well documented so far. Moreover, a high time resolution is required to analyze the ciliary movement of CPECs, in which expensive high-speed cameras are indispensable. To circumvent this necessity and simplify monitoring the ciliary motility of various cell types, a low cost, high-speed camera has been introduced, and an easily accessible and convenient method to record the motility of motile cilia, especially to describe the speed and pattern of motion of each cilium, has been developed1. Moreover, original image analysis software &#8220;TI Workbench&#8221; has been used here to facilitate detailed analysis of motility. Collectively, this method provides a new concise strategy to analyze ciliary motion together with correlative scanning electron microscopy (SEM), which can be adopted in a wide range of cilium research.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>for both live imaging and SEM preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereo microscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td></td>
		</tr>
		<tr>
			<td>flat paper towel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#934;10-cm plastic dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100-mL beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>straight operating scissors</td>
			<td>Sansyo</td>
			<td>S-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>watchmaker forceps</td>
			<td>Dumont</td>
			<td>No.DU-3 or -4, INOX</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass bottom dish</td>
			<td>Matsunami Glass</td>
			<td>D110300</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alminum foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-mL glass vial with a polyethylene cap</td>
			<td>Nichiden Rika-Glass</td>
			<td>PS-5A</td>
			<td></td>
		</tr>
		<tr>
			<td>transfer pipette</td>
			<td>Samco Scientific</td>
			<td>SM251-1S</td>
			<td>for specimen tranfer</td>
		</tr>
		<tr>
			<td>toothpick</td>
			<td></td>
			<td></td>
			<td>for specimen transfer</td>
		</tr>
		<tr>
			<td>ion sputter with gold-palladium</td>
			<td>Hitachi</td>
			<td>E-1030</td>
			<td></td>
		</tr>
		<tr>
			<td>critical point dryer</td>
			<td>Hitachi</td>
			<td>HCP-2</td>
			<td></td>
		</tr>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>100 W mercury lump housing and power supply</td>
			<td>Olympus</td>
			<td>U-ULH and BH2-RFL-T3</td>
			<td></td>
		</tr>
		<tr>
			<td>100 W mercury lamp</td>
			<td>Ushio</td>
			<td>USH103D</td>
			<td></td>
		</tr>
		<tr>
			<td>DIC condenser, n.a. 0.55</td>
			<td>Olympus</td>
			<td>IX-LWUCD</td>
			<td></td>
		</tr>
		<tr>
			<td>electrrical shutter</td>
			<td>Vincent Associates</td>
			<td>VS35S22M1R3-24 and VMM-D1</td>
			<td>manual shutter can be used.</td>
		</tr>
		<tr>
			<td>band-pass filter (400-700 nm, &#934;45 mm)</td>
			<td>Koshin Kagaku</td>
			<td>C10-110621-1</td>
			<td></td>
		</tr>
		<tr>
			<td>ND filter (&#934;45 mm)</td>
			<td>Olympus</td>
			<td>45ND6, 45ND25</td>
			<td>combination of 25% and 6% ND filters are used</td>
		</tr>
		<tr>
			<td>objective lens (water immersion) with DIC element</td>
			<td>Olympus</td>
			<td>UApo 40XW/340, n.a., 1.15 with IX-DPAO40</td>
			<td></td>
		</tr>
		<tr>
			<td>high-speed video camera</td>
			<td>Allied Vision Technologies</td>
			<td>GE680</td>
			<td>&#62;= 200 Hz frame rate and 1 msec expose time</td>
		</tr>
		<tr>
			<td>image acquisition / analysis software</td>
			<td>in-hous software</td>
			<td>TI Workbench</td>
			<td>capable of acquisition at high frame rates.</td>
		</tr>
		<tr>
			<td>PC for camera control / analysis</td>
			<td>Apple</td>
			<td>Mac Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>vibration isolation table</td>
			<td>Meiritsu Seiki</td>
			<td>AD0806</td>
			<td></td>
		</tr>
		<tr>
			<td>weight for tissue</td>
			<td>Warner Instruments</td>
			<td>slice anchor kits</td>
			<td>It can be made with nylon mesh glued to a U-shape squashed &#934;0.5mm platinum wire.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>scanning electron microscope</td>
			<td>JEOL</td>
			<td>JSM-6510</td>
			<td></td>
		</tr>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol&#160;</td>
			<td>Wako Chemicals</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz L-15 medium</td>
			<td>Life Technologies</td>
			<td>11415-064</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol&#160;</td>
			<td>Wako Chemicals</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Life Technologies</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde&#160;</td>
			<td>Merck</td>
			<td>1040051000</td>
			<td></td>
		</tr>
		<tr>
			<td>glutaraldehyde&#160;</td>
			<td>Nacalai tesque</td>
			<td>17003-05</td>
			<td></td>
		</tr>
		<tr>
			<td>isoamyl acetate</td>
			<td>Nacalai tesque</td>
			<td>02710-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Sieves 4A 1/8&#160;</td>
			<td>Wako Chemicals</td>
			<td>130-08655</td>
			<td>for preparation of anhydrous ethanol</td>
		</tr>
		<tr>
			<td>phosphate buffer saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffer, 0.1 M</td>
			<td></td>
			<td></td>
			<td>To make 100 ml, mix 19.0 ml of 0.1 M NaH2PO4 and 81.0 ml of 0.1 M Na2HPO4&#160;</td>
		</tr>
		<tr>
			<td>monosodium phosphate (dihydrate)</td>
			<td>Nacalai tesque</td>
			<td>31718-15</td>
			<td></td>
		</tr>
		<tr>
			<td>disodium phosphate (anhydrous)</td>
			<td>Nacalai tesque</td>
			<td>31801-05</td>
			<td></td>
		</tr>
		<tr>
			<td>suclose</td>
			<td>Nacalai tesque</td>
			<td>30406-25</td>
			<td></td>
		</tr>
		<tr>
			<td>osmium tetroxide</td>
			<td>Nisshin EM</td>
			<td align="right">300</td>
			<td></td>
		</tr>
		<tr>
			<td>dry ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

observation of the ciliary movement of choroid plexus epithelial cells ex vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

工作流程的概述示于图1，包括设备的图像。 CPECs活运动观测 电影1示出了电影CPECs从围产期小鼠中分离的，和电影2示出了在电影1的图像的放大图。应当注意的是，个人睫状提示是在与那些在电影相比静像不太清楚。 图2示出了跟踪两个纤毛与不同模式的运动，背面的往复运动和旋转运动，从...

Watch this Scientific Journal Video about Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo at JoVE.com

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

脉络丛上皮细胞的纤毛运动的观察<em&gt;离体</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

observation-ciliary-movement-choroid-plexus-epithelial-cells-ex

Research

JoVE Journal

Neuroscience

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

12.1K 次观看.  Waseda University. In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

视频: Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

体外器官角膜模型急性上皮疱疹，单纯疱疹病毒I型感染

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

科研

神经科学

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

糖尿病大鼠的热痛觉阈值的快速测定

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

前全体体内培养，发展的果蝇大脑

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

从大鼠脑的解剖脑表面血管及相关脑膜和脉络丛的可视描述，

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

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 ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

在体内 小鼠劣质橄榄中的钙成像

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

The present protocol describes the ex vivo calcitonin gene-related peptide (CGRP) release model and the strategy to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents.

离体 啮齿动物三叉血管系统中降钙素基因相关肽的释放

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in ...

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

大鼠坐骨神经用于 离体 神经生理学的制备

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...

脉络丛上皮细胞的纤毛运动的观察

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体外器官角膜模型急性上皮疱疹，单纯疱疹病毒I型感染

糖尿病大鼠的热痛觉阈值的快速测定

前全体体内培养，发展的果蝇大脑

从大鼠脑的解剖脑表面血管及相关脑膜和脉络丛的可视描述，

完整的犁鼻器和副嗅球的体外制剂

急性

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

在体内小鼠劣质橄榄中的钙成像

离体啮齿动物三叉血管系统中降钙素基因相关肽的释放

大鼠坐骨神经用于离体神经生理学的制备

脉络丛上皮细胞的纤毛运动的观察

摘要

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此视频中的章节

体外器官角膜模型急性上皮疱疹，单纯疱疹病毒I型感染

糖尿病大鼠的热痛觉阈值的快速测定

前全体体内培养，发展的果蝇大脑

从大鼠脑的解剖脑表面血管及相关脑膜和脉络丛的可视描述，

完整的犁鼻器和副嗅球的体外制剂

急性

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

在体内 小鼠劣质橄榄中的钙成像

离体 啮齿动物三叉血管系统中降钙素基因相关肽的释放

大鼠坐骨神经用于 离体 神经生理学的制备

在体内小鼠劣质橄榄中的钙成像

离体啮齿动物三叉血管系统中降钙素基因相关肽的释放

大鼠坐骨神经用于离体神经生理学的制备