我们要感谢他的援助，在视频编辑雅各索利斯。 这部分工作是由美国国家科学基金会职业奖DBI-0953902 MJ列文。

1。动物灌注5和整个小鼠脑结算<ol><li>的整个长度上的程序可以根据每脱水工序中使用的时间长度而变化，但总的整个过程可以在两天内进行。 </li><li>称重YFP的小鼠和然后深深麻醉腹膜内注射氯胺酮/赛拉嗪（100毫克/公斤10毫克/公斤）。 </li><li>确认的手术平面的深度麻醉，然后再进行手术。检查动物，每隔5分钟，看看它的反应到公司或脚趾尾捏。如果动物的反应，氯胺酮/赛拉嗪的补充剂量（原剂量的1/3）是必需的。 </li><li>一旦深度麻醉，抑制小鼠通过坚持每个肢体实验室磁带到手术床上使用，让鼠标是在仰卧位（背recumbancy），露出胸部手术。一张的手术通常是由一个金属或塑料网和放入水槽或顶部的唇形盘，使血液和固定液废物可以很容易地被收集。 </li><li>开始，一个低于剑突过程中的切口。剪切沿肋条笼形件的基础上用剪刀和镊子和回拉皮肤作为切割。削减沿鼠标胸骨两侧的（通过肋），创建一个皮瓣的组织，从胸腔内使用止血钳离开的心脏暴露。 </li><li>进入左心室的心脏插入了23 G针和一个小切口，右心房肌壁，使血液逃脱。请参阅朱庇特第2497此过程的视频。 </li><li>立即被切断后，右心房，开始灌注4℃的磷酸缓冲生理盐水，PBS（pH7.2）中，直到不再观察到血液的心脏的右心房（30 - 40毫升的速率约5从排水毫升/分钟）。 </li><li>一旦所有的血液被抽干（液体排出右心房是明确的），灌注介质切换到冷冻4％PFA解决方案。灌注，直到鼠标的身体变得明显的刚性和寒冷的触摸（约30 - 40毫升的速度约5毫升/分钟）。 （稀释16％集中PFA解决方案根据制造商的说明书中加入NaOH直至pH值达到7.2。戴上手套，实验室外套和处理，在通风橱内的化学品混合）。 </li><li>灌注后，移开鼠标，从开始的大脑切除的手术床上，斩首。 </li><li>使用镊子和虹膜剪，除去头骨，小部分从后面的头骨和前进的。小切口，用剪刀在整个头骨的2 - 4毫米，而使用镊子小心地将骨远离大脑中的小部分。做到这一点，直到整个顶表面被暴露的大脑。 </li><li>海关的大脑，头骨，5毫米宽，平锅铲和一个玻璃小瓶。淹没在4％PFA 6小时，在4℃下进行后固定。 </li><li>经过后期固定术，两次在室温PBS洗大脑通过浇注的煤灰，满分的玻璃小瓶中，并用PBS代替。涡流的大脑浇筑前的第二次洗涤和更换PBS PBS溶液。 </li><li>脱水的大脑进行梯度系列的乙​​醇孵育（一次在50％，70％，95％，和两次在100％）在2小时，每培养，然后为第二个的100％乙醇孵育12小时，在室温下通过提取的水，从固定的组织。每个孵化，以前的乙醇溶液倒入玻璃小瓶，直到大脑完全被水淹没，取而代之的是在随后的解决方案。 </li><li>在100％乙醇中的第二次温育后，倾出溶液，并更换与等份的乙醇和结算含有苄醇，苯甲酸苄酯（1:2体积：体积比）的溶液。经过2小时的潜伏期，滗析这个解决方案中，和取代苄醇，苯甲酸苄酯（1:2体积：体积比）以100％的溶液。该refra的莫如指数的结算解决方案，为n = 1.54。 </li><li>在巴布后，大脑会明显变得透明，在4 - 5小时。为了获得最佳的结算结果，离开大脑，以清除在室温下6天，同时屏蔽从明亮的光线。 </li></ol> 2。显微镜设置<ol><li>一旦大脑被清零，并准备用于成像，加盖的陪替氏培养皿的底部，使用氰基丙烯酸酯。使粘合剂干燥，然后再继续。 </li><li>干燥后，淹没巴布大脑中，用于成像的显微镜物镜下放置在陪替氏培养皿。 </li><li>我们使用了多光子显微镜，结合埋汰钛蓝宝石激光器710 nm至990 nm激发波长可调。我们用来生成YFP信号的激发波长为886纳米。激光功率范围从30 - 100毫瓦，根据成像深度。 </li><li>捕捉反射的荧光信号使用的是尼康5X物镜（NA 0.5），允许大字段的视图成像（2×2毫米）。 </li><li>过滤器反射的荧光信号使用一个五十〇分之五百三十五带通滤波器和收集它使用砷化镓PMT（H7422PA-40，滨松，布里奇沃特，新泽西州）。 </li><li>处理图像在ScanImage软件6，分辨率为2048×2048像素，扫描速度为2毫秒，每行生成高分辨率，YFP图像。 </li><li>一次成像完成后，消除大脑从培养皿中，使用产钳和存储在巴布避光未来的成像。对于胶样品，通过运行一个刀片的角度不大于30度之间的胶水和陪替氏培养皿中除去组织。我们已保存的样品在巴布长达1年没有明显的退化。 </li></ol> 3。代表性的成果此处所示的代表图像和视频显示的高分辨率的多光子成像能力成为可能的光透明。全脑成像允许YFP标记在不同的层，海马和皮层神经细胞清晰可见深组织表面的2毫米以下。 图1显示了一个代表图像的冠状相应的解剖学特征的海马2.92毫米尾鳍前囟门7。样品中的成像的深度为1.94毫米。这种差异是由于在尾端的大脑切除小脑的平衡，是由于脱水过程中收缩。从堆栈中的另一图像显示层V / VI下方的组织表面（ 图2）的新皮层表达YFP2毫米的锥体神经元。所有的图像被收购使用的尼康5X目标和放大倍率，使用数码变焦功能，图像采集ScanImage软件。 通过放大在新皮层中，明确个人在新皮层V层锥体神经元的轴突和神经元胞体光年区分至1.02毫米的组织表面的下方（ 图3）。从图3中使用的图像堆栈，神经元的区域的3D重建使用ImageJ软件8（ 图4）。 高分辨率的能力，也让我们的MPM图像的整体，单个神经元。在这里，我们展示的是重建的V层锥体神经元的大脑新皮质中，个体的树突清晰可见（ 图5）。 <img alt="图1" src="/files/ftp_upload/3848/3848fig1.jpg" /> 图1示出新皮质和海马的不同层的整个小鼠脑的代表图像从一个1.2毫米的图像堆栈（0.8 -下方组织表面2毫米）。特点是可见的海马组织表面的1.1毫米以下，并已使用如下标记ING代码：DG =齿状回，GrDG =颗粒层DG，Lmol的腔隙moleculare层，MOL =分子层DG，或= oriens的层，PoDG多形层DG，PY =锥体细胞层，抗辐射层radiatum。图片是1.8×1.8毫米大小，比例尺= 200微米。的脑吻侧端被固定到与尾端朝上朝向目标陪替氏培养皿。这有助于在冠状面成像。 <img alt="图2" src="/files/ftp_upload/3848/3848fig2.jpg" /> 图2。代表整个小鼠脑组织突出新皮层1.2毫米的图像堆栈帧（0.8 - 2毫米的组织表面下）。锥体细胞和表达YFP V / VI的皮质层是可见的组织表面的2毫米以下。程度与以前的报告在新皮层稀疏的标签，标签上是一致的。9插图（右）从B扩大<html>上左边的oxed地区。图片的尺寸为1.8×1.8毫米，比例尺= 200微米（50微米插图）。 <img alt="图3" src="/files/ftp_upload/3848/3848fig3.jpg" /> 图3。代表帧V层锥体神经元的大脑新皮质中从图像堆栈下方的组织表面774微米。堆栈从700微米到1020微米的组织表面的下方。一个8倍数码变焦被用来捕捉细节，如精细的工序。图片为225×225微米大小，比例尺= 20微米。 <img alt="图4" src="/files/ftp_upload/3848/3848fig4.jpg" /> 图4代表三维重建图像（225×225×320微米），在图3中的图像堆栈。图片为225×225微米大小，比例尺= 28微米。 的“&gt; <img alt="图5" src="/files/ftp_upload/3848/3848fig5.jpg" /> 图5。高分辨率重建图像的V层锥体神经元的新皮层使用10倍数码变焦。神经元尺寸为485微米长，比例尺= 25微米。为每个图像平铺的视场为180微米×180微米。每一瓦片是在一个不同的深度（z轴的水平）。皮质顶树突不这样做，在一般情况下，快递YFP以及胞体。都在同一视野，如下图所示，电源降低到最小化的胞体的饱和度，这使得精细工艺的外观调光器。我们这里强调的是在展示场的看法，而不是精细的细节。为了显示更精细的细节，使用数码变焦，集中在一个地区，而胞体，并提高激光功率。

<ol>
	<li>Parra, S. G., Chia, T. H., Zinter, J. P., Levene, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+microscopy+of+cleared+mouse+organs.">Multiphoton microscopy of cleared mouse organs.</a> J. Biomed. Opt. 15, 036017 (2010).</li><li>Vesuna, S., Torres, R., Levene, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+fluorescence,+second+harmonic+generation,+and+fluorescence+lifetime+imaging+of+whole+cleared+mouse+organs.">Multiphoton fluorescence, second harmonic generation, and fluorescence lifetime imaging of whole cleared mouse organs.</a> J. Biomed. Opt. 16, 106009 (2011).</li><li>Zucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+insect+and+mammalian+embryo+imaging+with+confocal+microscopy:+Morphology+and+apoptosis.">Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis.</a> Cytometry. A69, 1143-1152 (2006).</li><li>Sakhalkar, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+in+bulk+tissue+specimens+using+optical+emission+tomography:+Fluorescence+preservation+during+optical+clearing.">Functional imaging in bulk tissue specimens using optical emission tomography: Fluorescence preservation during optical clearing.</a> Phys. Med. Biol. 52, 2035-2054 (2007).</li><li>Dazai, J., Spring, S., Cahill, L. S., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-mouse+Neuroanatomical+Magnetic+Resonance+Imaging.">Multiple-mouse Neuroanatomical Magnetic Resonance Imaging.</a> J. Vis. Exp. (48), e2497 (2011).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomed. Eng. Online. 2, (2003).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Abramoff, M. D., Magelhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Feng, G., Mellor, R. H., Bernstein, M., Keller-Peck, C., Nguyen, Q. T., Wallace, M., Nerbonne, J. M., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Neuronal+Subsets+in+Transgenic+Mice+Expressing+Multiple+Spectral+Variants+of+GFP.">Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Hama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat. Neuro. 14, 1481-1488 (2011).</li></ol>

按照耶鲁大学实验动物护理和使用委员会提出的指导方针和规定，对动物进行的实验。

虽然标准的有机染料是兼容的有机溶剂的范围内，因此，不清除协议构成特别的挑战，荧光蛋白往往是在溶剂中的改变的耐性低。4本工作的目的是克服一个严重的限制以前的光学结算协议XFPS荧光的丢失或严重退化。这里所呈现的图像表明，荧光YFP被保存。光透明这里描述的技术允许YFP标记的神经元在长达2毫米的整个鼠脑组织表面下的高清晰度成像。先前证明，使用乙醇脱水，也将保留?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td>磷酸盐缓冲液</td><td> Sigma-Aldrich公司</td><td> D8537 </td><td> 500毫升，pH 7.2的</td></tr><tr><td>多聚甲醛</td><td>电子显微镜科学</td><td> 15710 </td><td> 10×10毫升，16％多聚甲醛</td></tr><tr><td>乙醇</td><td>美国生物分析</td><td> AB00515-00500 </td><td> 500毫升，200证明</td></tr><tr><td>乙醇</td><td> Pharmco产品，Inc。 </td><td> 111000190 </td><td> 1加仑，190证明</td></tr><tr><td>苯甲醇</td><td> Sigma-Aldrich公司</td><td> 402834 </td><td> 500毫升，99 +％的</td></tr><tr><td>苯甲酸苄酯</td><td> Sigma-Aldrich公司</td><td> B6630-IL </td><td> 500毫升，≥99％的</td></tr><tr><td> 5X/0.5 NA目标</td><td>尼康</td><td> AZ计划面粉5倍</td><td> 15 WD </td></tr></tbody></table>

multiphoton microscopy of cleared mouse brain expressing yfp

多光子显微镜是通过光学成像之前清除器官的固有荧光和二次谐波产生（SHG）的整个鼠标机关。1,2然而，对于器官，包含荧光蛋白，如GFP和YFP，光使用甲醇的结算协议脱水和用苄醇苯甲酸苄酯（巴布），而未受保护的光3不保留的荧光信号。这里介绍的是一种新的方式，同时保留YFP的荧光信号在神经元中表达，在其中执行整个器官对小鼠脑组织的光透明的协议。改变光透明的协议，例如使用的乙醇梯度系列已经发现，减少损伤的荧光蛋白，并保持其多光子成像的荧光信号，器官是脱水。4使用一个优化的方法，光透明的基于乙醇的脱水和清除由巴布从光屏蔽时，我们显示高分辨率的多光子图像，黄色荧光蛋白（YFP）表达在神经元的小鼠脑组织表面下方超过2毫米。

Watch this Scientific Journal Video about Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP at JoVE.com

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

整个小鼠脏器的多光子显微镜能够通过光学成像之前清零器官，但并非所有协议保留的荧光蛋白的荧光信号。使用光学乙醇脱水和苯甲醇苯甲酸苄酯结算的结算方法，显示高分辨率的多光子图像的整个小鼠脑组织表达YFP。

多光子显微镜的清除小鼠脑组织表达YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

multiphoton-microscopy-of-cleared-mouse-brain-expressing-yfp

Research

JoVE Journal

Neuroscience

13.8K 次观看.  Yale University. 整个小鼠脏器的多光子显微镜能够通过光学成像之前清零器官，但并非所有协议保留的荧光蛋白的荧光信号。使用光学乙醇脱水和苯甲醇苯甲酸苄酯结算的结算方法，显示高分辨率的多光子图像的整个小鼠脑组织表达YFP。

视频: Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

制备小鼠脑组织中的免疫电镜

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

科研

神经科学

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

聚焦离子束铣削和脑组织的扫描电子显微镜

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...