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><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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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 (18 x 18)</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&amp;nbsp;</td><td>Nikon TE2000</td><td>60X oil&amp;nbsp;</td></tr><tr><td>Microscope cover glass 24 x 60 mm&lt;sup&gt;2&lt;/sup&gt;</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

13.8K 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

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

科研

神经科学

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 ...

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 ...

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.

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. ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

可视化发育中的小鼠大脑中的神经元和神经胶质细胞动力学

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

作者聚焦：探索发育中的大脑中的细胞迁移和基因作用

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

室管膜纤毛在小鼠脑侧脑室实时成像

摘要

探索更多视频

此视频中的章节

体外实时成像在小鼠神经上皮单细胞分裂

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

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

脉络丛上皮细胞的纤毛运动的观察

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像