Authors would like to thank Charisse Montgomery for her editing service. A. Alomran&#8217;s work partially fulfills the requirements for a master&#8217;s degree in Pharmacology.This work is funded by The University of Toledo&#8217;s intramural startup fund for W.A.A and NIH grant (DK080640) for S.M.N.

该程序使用的动物均按照该机构动物护理和使用委员会的指导方针，在卫生部和国家研究院​​的指南护理和使用经托莱多大学的机构动物护理和使用委员会（IACUC）实验动物。 1.脑提取，切片和组织准备<ol><li>通过用CO 2窒息深深安乐死5分钟牺牲野生型小鼠品系C57BL / 6。颈椎脱位保证死亡。 </li><li>清洁鼠标头，用70％的乙醇。 </li><li>由第一牵引皮肤脱落，开始与头部的顶部，以暴露颅骨执行使用无菌剪刀和镊子骨瓣。 </li><li>然后，当颅骨露出，通过剥离骨片逐片，从后侧开始并朝着前侧移除头骨。要小心，不要破坏脑室。 </li><li>收集整个大脑。 </li><li>将脑在含有DMEM /高葡萄糖补充有10％胎牛血清（FBS）和1％青霉素/链霉素溶液含有青霉素10000单位/毫升，链霉素和预热10,000微克/毫升100毫米的培养皿至37℃。 </li><li>切片上的正中矢状面的脑用手用锋利的刀片，并获得使用振动的每个半第一100-200毫米截面。 </li><li>冲洗脑组织与预热37℃的磷酸盐缓冲盐水（1×PBS）中的溶液。 </li><li>立即将在DMEM /高葡萄糖培养基预温热至37℃的脑切片。 </li></ol> 2.实时成像配置和设置<ol><li>将脑组织切片中含有1ml的DMEM /高葡萄糖培养基的30mm的玻璃底培养皿。调整显微镜的封闭室的环境中37℃，95％/ 5％的O 2 / CO 2的含量（ 图1 </str翁&gt;）。 </li><li>使用60X物镜油浸镜头，首先将在60X物镜的油滴，并专注于细胞定期DIC收集室管膜细胞/纤毛的图像传输的光。 </li><li>然后，按照DMEM气泡运动作为对能动的纤毛室管膜的位置的引导方向为纤毛殴打建立在该地区的一种气泡的运动。选择含有健康的细胞，在使用DIC过滤大脑的侧脑室动纤毛的区域。一旦室管膜纤毛被发现，调节光线和聚焦，以获得令人满意的图像。 </li><li>根据使用的Metamorph成像软件的特定目的设定实时成像参数。在本演示中，获得24位图像用相机分级设置为1×1结合60X客观和5-10毫秒的曝光时间。 </li><li>通过以具有最小EXP打开显微镜孔径到最佳水平收集的DIC图像osure时间。观察实时图像流的摄像头，提供快速，即时的图像采集刻不容缓。计算纤毛跳动基于最小曝光时间的要求，以获得足够的图像对比度的速度。 </li></ol> 3.数据可视化和分析<ol><li>通过计数1分钟殴打的数量计算纤毛殴打的数量。通过减小视频的速度和计数使用细胞计数器或类似工具的节拍数要这样做。 </li><li>为了计算殴打的频率，乘以在该视频记录被获取以获得秒的数目的帧或时间推移的图像的数量的曝光时间。 （实施例：曝光时间5毫秒200帧= 1000毫秒或1秒）。 </li><li>计算在一秒钟的殴打的数量，以获得被表示为每秒跳动的次数的频率。通过将纤毛殴打的人数超过一秒钟的时间跨做到这一点VAL（例：纤毛跳动50次的200帧视频录制，在5毫秒即 5毫秒×200帧= 1秒的曝光时间，现在分50次1秒= 50赫兹）。 </li><li>通过在电源和恢复招评价都采取了室管膜纤毛的路径计算纤毛跳动角度。根据先前描述的方法执行此，具有小的修改11。个别纤毛的精确运动在完整周期节拍观察。 </li><li>关于乙酸酯片材放置在显示器上，绘制一条水平线沿室管膜边缘并通过纤毛在做功冲程开始时的中线位置的垂直线。 </li><li>由帧绘制纤毛帧的精确位置，因为它向前移动时在做功冲程期间。以类似的方式，绘制恢复行程中纤毛的运动。 </li><li>从T期间中线计算从纤毛的最大偏差睫状打浆角他动力冲程以及恢复行程。 </li></ol> 4.钙信号记录<ol><li>切片大脑后，短暂地冲洗的脑切片用1×PBS或Dulbecco氏PBS（pH7.0）中。制备新鲜的Fluo-2，以避免荧光猝灭，并获得良好的信噪比。 </li><li>制备的Fluo-2溶液在二甲基亚砜（DMSO）的1mM储备溶液，混合并旋涡振荡溶液至少5分钟，以确保将Fluo-2被均匀溶解在DMSO中。 </li><li>稀释的Fluo-2原液在500毫升的DMEM /高葡萄糖的补充有2％B27的预加热到37℃至20毫克/毫升的最终浓度。 注：B-27是一种含有维生素A，抗氧化剂鸡尾酒，用于支持海马及其他CNS神经元的短期或长期生存能力胰岛素优化的无血清补充。 </li><li>立即孵育大脑切片用20毫克/毫升的Fluo-2进行30分钟，在37℃，在玻璃底板。 </li><li>为了确定钙荧光团的Fluo-2的最佳负载浓度并避免钙染料细胞毒性，与ATP挑战细胞，并通过确定响应于ATP的时间过程和钙信号的峰值幅度检查细胞存活力。 </li><li>记录为钙振荡视频以5毫秒的最小1秒（每秒200帧）的捕获率，用488纳米和515纳米，分别（ 电影2）的激发和发射波长。 </li><li>要区分自体荧光或运动伪影的荧光 - 2钙信号，保证在515纳米发射的强度进行独立监控。 注：将Fluo-2不适合于量化细胞内钙的钙荧光光度法。然而，这是一个极好的动态钙染料检测钙的快速变化，如钙振荡。为了更准确的定量的钙，呋喃-2建议。然而，的Fura-2的动态变化是由它的比例限定染料的性质。 </li><li>遵循由生产商提供的公式来计算的确切细胞内游离钙的值。例如钙离子浓度= K 深 ×（R - R最小值）/（R 最大 - R）。其中，K d为从释放钙染料的解离常数，R为所测量的荧光在488和R 最小和R 最大值是在最小和最大离子浓度12的荧光比率。 </li></ol> 5.免疫荧光显微<ol><li>固定的脑切片用含有4％多聚甲醛（PFA）和2％的蔗糖，10分钟的磷酸盐缓冲盐水溶液。可替代地，固定的全脑，用4％PFA再部成使用低温恒温器50毫米部分。 </li><li>用1×PBS，每次5分钟，洗净组织三次。 </li><li>孵育大脑切片用soluti上0.1％的Triton-X在1×PBS中5分钟，再漂洗三次用1×PBS，每次5分钟。 </li><li>孵育脑切片与小鼠抗，antiacetylated一个微管蛋白，用在稀释1：5000，在10％FBS的1×PBS在室温（RT）或过夜，在4℃一小时。 </li><li>用1×PBS，每次5分钟，洗净组织三次。 </li><li>孵育在二级抗体的脑切片，荧光素的抗小鼠IgG的稀释度1：在1小时在室温下的1×PBS溶液500在10％FBS。 </li><li>在荧光显微镜下观察，染液的部分用4&#39;，6-二脒基-2-苯基吲哚（DAPI）中5分钟，进行染色的核（或DNA）13之前。以最小的曝光时间可能立即章节尽量减少漂白，形象。 </li></ol>

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没有利益冲突的声明。

这里描述的是协议用于制备鼠脑组织为活的成像和荧光显微镜，提供了脑室内的快速，灵敏密切观察室管膜纤毛。这种技术不限于仅侧脑室;它可以被用来观察在其他脑室的纤毛。这种成像技术提供的实时流类似于在离体环境的脑脊液由纤毛跳动的运动。此外，它使纤毛的定向运动的分析。这主要是促进通过使用高分辨率DIC和荧光成像系统。这个系统的一个优点是，在显微镜被封闭在一个环...

纤毛是感官微管系的细胞器，从细胞表面延伸到细胞外环境。取决于它们的微管组织，纤​​毛可分为两种类型 - “9 + 0”或“9 + 2”。在功能上，根据他们的运动，这些可被归类为运动型或非运动纤毛1。原发性纤毛是一种常用来表示“9 + 0”不动纤毛的术语。这些有九并行双峰微管（记为&#39;9&#39;）和一个中心对微管是中央鞘（表示为&#39;0&#39;）内不存在。然而，一些“9 + 0”的纤毛，如节点纤毛，从而调节胚胎偏侧是能动2。另一方面，运动纤毛的特征在于，除了九个并行微管双峰，通过附加中央对微管双峰，并与动力蛋白马达蛋白相关联，以促进蠕动。另外，一些“9+2“纤毛等纤毛的嗅觉都是非能动3。室管膜细胞衬脑室和脊髓的中央管的特征在于运动纤毛该推动脑脊液（CSF）沿脑室4。 该方法的总的目标是促进学习运动纤毛动力学和结构异常。大脑的健康和发展严重依赖于脑脊液的脑室中的高效流通。例如，正常脑脊液流量和流体平衡需要正常跳动和功能室管膜纤毛5,6，进而在调节神经元细胞的定向运动发挥关键作用和干细胞迁移7。因此，异常的室管膜纤毛功能或结构可能会导致异常脑脊液流动，这与脑积水，医疗条件，其中有脑脊液中的b的心室的异常积累雨。这可能进而使得颅内压增高和头部，抽搐，隧道视野的逐步扩大，以及精神残疾8。 该技术优于现有方法的优点是，它允许首次报道三个不同的室管膜细胞类型：I，II和III，基于其独特睫状打浆次数跳动角度。这些室管膜细胞在脑室某些区域内本地化。此外，年龄和药理学试剂如酒精和西洛他唑在改变室管膜细胞类型或它们的本地化的影响可以证明，这是不可能的这种分类室管膜细胞之前。西洛他唑是磷酸二酯酶3抑制剂，即代谢的cAMP为AMP的酶，它也调节细胞内钙9。采用高速荧光成像分析，可以成像的独特细胞内和细胞量化室管膜细胞的钙振荡特性。例如，酒精和西洛他唑显著改变室管膜纤毛跳动频率以及细胞内钙振荡性能，这反过来可能导致由室管膜纤毛10在脑脊液体积置换的变化。总之，该技术是关键，以提供三种不同类型的室管膜细胞具有不同钙振荡特性的第一个证据。 在下面的章节中，程序的详细一步一步的概述提供，密切关注组织准备和处理到。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/HIGH GLUCOSE</td>
			<td>Cellgro Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30088-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermo Scientific</td>
			<td>SH30256-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S-2395</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-2&#160;</td>
			<td>TEF Labs</td>
			<td>#0200</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD HardSet Mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetylated a-tubulin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T7451&#160;</td>
			<td>clone 6-11B1</td>
		</tr>
		<tr>
			<td>FITC Anti-mouse antibody</td>
			<td>Vector Labs</td>
			<td>FI-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture plate</td>
			<td>VWR Vista Vision</td>
			<td>30-2041</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slip (18x18)</td>
			<td>VWR Vista Vision</td>
			<td>16004.326</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems</td>
			<td>Leica VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td>Leica CM1860</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>Nikon&#160;</td>
			<td>Nikon TE2000</td>
			<td>60X oil&#160;</td>
		</tr>
		<tr>
			<td>Microscope cover glass 24x60mm</td>
			<td>VWR Vista Vision</td>
			<td>16004-312</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI filter cube</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of the ependymal cilia in the lateral ventricles of the mouse brain

Multiciliated室管膜细胞排列在成人大脑脑室。功能异常或室管膜纤毛的结构与各种神经功能障碍有关。目前能动纤毛室管膜技术的体外实时成像允许纤毛动态以下几个步骤进行详细的研究。这些步骤包括：小鼠安乐死二氧化碳根据托莱多的机构动物护理和使用委员会（IACUC）的大学协议;骨瓣其次为脑拆除和矢状脑解剖与vibratome或锋利的​​刀片通过大脑侧脑室，其中室管膜纤毛可以可视化，以获得非常薄的部分。大脑的切片在含有Dulbecco氏改良的Eagle氏培养基（DMEM）/高葡萄糖，在37℃在95％/ 5％氧气的存在下2 / CO 2混合物定制玻璃底板孵育必须保持所述组织活中实验。纤毛跳动的视频然后，使用高分辨率微分干涉显微镜记录。则视频被分析帧由帧来计算纤毛跳动频率。这允许基于其纤毛跳动频率和角度的室管膜细胞为三类或类型的不同分类。此外，这种技术允许使用高速荧光成像分析以表征室管膜细胞的独特细胞内钙振荡特性以及药理学试剂的钙振荡的效果和睫状跳动频率。此外，该技术适用于免疫荧光成像纤毛结构和纤毛蛋白定位研究。这是在疾病的诊断和表型的研究尤为重要。该技术的主要局限性是由于在活动纤毛运动的减少作为脑组织开始死亡。

在活老鼠大脑室管膜下的测量功能纤毛在这个协议中所描述的方法被用于监控在新鲜组织从小鼠脑以及监测和研究纤毛跳动频率解剖室管膜纤毛功能和结构。接着完成一个完整的实验步骤描述于一个概要流程图（图1）。强烈建议在实验很短的时间内，以保持运动纤毛尽可能积极进行。代表时间推移电影和室管膜细胞和它们的运动纤毛的图像也显示（电影1 <e...

Watch this Scientific Journal Video about Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain at JoVE.com

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

使用高分辨率微分干涉对比（DIC）显微镜， 离体观察位于小鼠脑室内游动室管膜纤毛的跳动的证明通过实时成像。该技术允许独特纤毛跳动频率的记录和跳动角度以及它们的细胞内钙振荡起搏属性。

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

live-imaging-ependymal-cilia-lateral-ventricles-mouse

Research

JoVE Journal

Neuroscience

13.6K 次观看.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. 使用高分辨率微分干涉对比（DIC）显微镜， 离体观察位于小鼠脑室内游动室管膜纤毛的跳动的证明通过实时成像。该技术允许独特纤毛跳动频率的记录和跳动角度以及它们的细胞内钙振荡起搏属性。 

视频: Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

科研

神经科学

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

手动排水的斑马鱼胚胎脑室

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

在小鼠大脑的各个区域端粒酶活性：非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

脑室周围组织的侧脑室及组织学特性对人类和鼠标3D建模

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

探索深空-揭开人脑侧脑室的脑室结构解剖

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.