This work was supported in part by National Institutes of Health (NIH) Grants R01 GM-41514 (to M.D.C.), R39 GM-048071 (to I.P.), and R01 NS-074987 (to T.E.L.) and the National Multiple Sclerosis Society (NMSS) Collaborative Center Research Award CA1058-A-8 (to C.M.W., T.E.L. and M.D.C.), NMSS Grant RG4925, NIH Training Grant T32-AI-060573 (to M.L.G.), NMSS Postdoctoral Fellowship FG 1960-A-1 (to J.G.W.), and funding from the George E. Hewitt Foundation for Medical Research (M.P.M.).

注:伦理学声明:本协议动物处理批准了加州大学尔湾分校，协议＃2010至2943年的大学机构动物护理和使用委员会（IACUC）。 1.去除脊髓<ol><li>将浸湿〜100％的液态异氟烷，USP纸巾室实施安乐死，并把干燥​​的纸巾之上。鼠标放置在室内的干纸巾顶部，这样鼠标没有接触异氟醚，并确保室内覆盖。等待呼吸停止后至少一分钟，以确保鼠标安乐死。 </li><li>执行颈部脊髓横断确保鼠标安乐死。不要执行颈椎脱位，因为这可能损伤脊髓。 </li><li>喷小鼠用70％乙醇润湿的头发上。 </li><li>用锋利的剪刀精去除头发和皮肤从鼠标的背面脊柱约颈椎椎板1暴露（C1）至骶薄片4（S4）。 </li><li>使用手术刀用＃10刀片，使切口到脊柱到它从肌肉和脂肪中分离的左边和右边。这将使除去脊髓容易得多。 </li><li>可选:弯曲的鲁尔咬骨钳可以用来舀走更多的肉。 </li><li>同时保持椎骨与锯齿格雷夫钳的顶部，将钛弯曲Vannas剪刀弯侧成在C1暴露脊柱，小心不要碰到脊髓。 </li><li>滑动钛弯曲Vannas剪刀一路的权利，做一个小切口。小紧缩的声音应该被听到和感受到的剪刀剪的椎骨。上线的最左侧重复此削减。要小心，不要去得太快或进行过大的削减，因为这会冒险用剪刀损坏电源线。 </li><li>单个薄片应该能够解除与镊子一旦椎骨的两侧被切除。 řEPEAT每个层直到S4这一步，继续保持和拉回来脊髓椎骨。 注:在移植部位上方的椎骨将可能脱落，一旦它被砍倒在移植部位。继续首发略低于移植部位的程序。 </li><li>可选:打好椎骨下来，看看那里的移植部位和措施，削减〜20毫米喙和尾椎移植部位。 注意:当移栽的NPC多将不再需要作为移植的NPC一般不更远大于15mm迁移。需要脊髓的量将依赖于移植的细胞类型的实验和迁移特性。使延髓和尾鳍的削减是等距离的移植部位将使移植部位进行成像过程中更容易找到。 </li><li>使用手术刀用＃11刀片小心切开神经节的右侧和脊髓起始于喙端部的腹侧的左边。该W生病使脊髓从腹侧椎骨更容易地除去。 注:快速完成（步骤1.7-1.11），脊髓不会干。然而，1×PBS中可施用到帘线以保持湿润。 </li><li>反转鼠标按住鼠标了这么脊髓朝下。采用封闭式锯齿格雷夫钳小心剥离出脊髓。无切口脊髓，小心切开任何剩余神经节，以使脊髓在单个完整片被提起。 </li><li>确保脊髓在RPMI-1640，并把它放在冰上运输到2P显微镜。 </li></ol> 2.准备脊髓的成像<ol><li>嵌入在琼脂糖脊髓<ol><li>保持孤立的脊髓冷冻RPMI-1640的前成像冰。 </li><li>称出琼脂糖（低胶凝温度），并在5毫升1×PBS中制备5％的溶液。微波的5％的琼脂糖溶液，持续15秒，以溶解股份公司站起身来，让解决方案冷却到37℃。 </li><li>通过将其放置在封口膜的片材与腹侧朝上制备脊髓。 </li><li>吸管约5毫升在脊髓的腹侧5％琼脂糖溶液。让琼脂糖通过冷却到室温固化。充分凝固需要大约5分钟。 </li><li>涂上一薄层组织粘合剂到22平方毫米的盖玻片。 </li><li>反转嵌入脊髓，将腹侧上的封口膜。背侧现在将朝上。坚持盖玻片的背侧，并淹没琼脂糖嵌入脊髓/盖玻片准备在RMPI固化胶粘剂。去除多余的凝固用刀片琼脂糖。 </li></ol></li><li>在显微镜舞台上安装的脊髓<ol><li>适用凡士林至盖玻片的底部。这将稳定灌注在准备。 </li><li>将脊髓制备在一个以及成像显微镜舞台上与腹侧朝上向着25X浸渍目标。所述定制成像井是约20毫米深，50mm长，和50mm宽。 </li><li>图像，同时灌流与温热的制备（37℃），氧化（95:5氧气:二氧化碳卡波金）的RPMI-1640培养基中无血清。 <ol><li>预暖媒体到37℃的水浴中，并将其通过将管导入介质瓶连接到管道泵。 </li><li>保留介质温度在37℃的加热装置在所述管道的所述腔室的连接。 Superfuse媒体通过井以3毫升/分钟使用管连接到所述管式泵（ 图1B）。 </li></ol></li></ol></li></ol> 3. 2P成像脊髓腹侧的<ol><li>显微镜设置<ol><li>从获得使用变色龙超钛大鼠Thy1-EYFP脊髓图片:蓝宝石激光调谐到900纳米。衰减激光功率的标本&lt;5％，以确保最小的光毒性16。维持温度通过使用单一的直列加热器溶液，以确保稳定的成像，并以防止组织漂移和细胞损伤灌注氧灌注的RPMI-1640以恒定37℃。 </li><li>分离绿色荧光蛋白和EYFP荧光信号，将一个520纳米的单刃分色和560 nm的单刃分色分束器串联分离2P排放分为三个通道，故意分裂绿光发射，以提高知名度。 注意:这些信道被称为蓝绿色（发射&lt;520纳米），黄绿色（520-560纳米），和红色（发射&gt; 560纳米）。光电倍增管检测在每个通道发射的光。 </li></ol></li><li>感兴趣的定位成像领域<ol><li>使用目镜和亮场光源，聚焦浸渍物镜在脊髓设置一个参考点的腹侧边缘。 </li><li>确保环境光源熄灭和SW痒2P激发通过打开激光快门。 </li><li>如果可用，对于感兴趣的领域搜索组织时，使用较低的分辨率设置，不使用数码变焦更高的容量，并获得最终的图像时高于扫描速率。与感兴趣区域寻找时，组织本系统的典型设置为:分辨率:256像素;体积:X = 600微米，Y = 600微米，Z = 0-300微米。 </li><li>观察邻近脊髓的腹侧边缘EYFP轴突。由胶原（蓝色）的第二次谐波信号将是最亮的脊髓组织边缘。定位EYFP轴突只是背侧到二次谐波信号来自胶原和EYFP信号可视化的黄绿色通道。 </li><li>定位移植部位在脊髓制备14的纵向中心。对于小鼠1天以下EGFP-NPC移植，通过聚焦在z平面光路深入到组织中找到移植部位。移植部位的WiLL略有不同动物之间，但一般位于〜200μm的从腹侧边缘。观察EGFP-的NPC的集群在白质，接近腹部和侧边缘在小鼠体内后第1天，移植后。 </li></ol></li><li>获取最终图像<ol><li>收购512像素的图像分辨率;体积:X = 270微米，Y = 212微米，和z = 100微米使用Slidebook 6软件。在2.5微米的增量捕捉连续焦平面编译Z-栈。 </li><li>用适当的交错偏移，以确保在脊髓任何迁移对象的快速定量执行双向扫描。确保了理想的探测帧速率，以确定蜂窝速度内的脊髓约为1帧/秒。 注:成像设置可以改变单个用户偏好，诸如探测帧速率，图像分辨率和成像卷。更大的成像量一般会需要更长的时间来获得在GIV恩分辨率。 </li><li>分析，作物，光滑，伪Slidebook图像与图像分析软件，如位面了Imaris 7.7文件。通过梳理使用Slidebook和了Imaris一个自动化的过程连续成像体积产生最后的时间推移录像。 </li></ol></li></ol>

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The authors have no competing interests.

实时2P成像完好的组织要求调查NPC动力学和相互作用下移​​植到脱髓鞘小鼠脊髓。活2P成像通常用于确定在活的小鼠上脊髓背侧细胞动力学，并已经被用于研究背侧脱髓鞘在脱髓鞘疾病17-19。但是，因为移植的NPC迁移到腹侧白质，使用2P显微镜其位于太深图像在原位， 离体的准备是必要的。这一方法已被用于在我们的实验室确定的NPC移植到受损的和未受损脊髓6的能动性和髓鞘再生动力学。 体外制剂启用扫描脊髓不仅纵向，而且横向从腹侧6,20 。这允许对在吨在细胞运动性（速度和方向），并相互作用分析ransplant部位，相邻的移植位点，并在从移植位点6的各种距离。而我们最初设计该制剂将图像的移植EGFP-NPC的对脊髓的腹侧，它可以用于图像的任何荧光标记或从腹侧，背侧染色细胞，或横侧通过改变取向的脊髓安装。该制剂在活体成像的好处包括允许腹侧成像和缺乏需要麻醉和存活手术，这仍需要使用一个&#39;玻璃窗&#39;设置了允许多个成像会话的新活制剂，而不重复手术19,21 。应当注意的是，此过程相比，与活体成像组合了"玻璃窗"的方法的一个限制是不能从单个小鼠获得多个时间点，由于脊髓被去除，小鼠被施以安乐死。 2P成像非常适合于解决完整的组织在22,23单个细胞的动态。 2P激发利用近红外光子，允许对深部组织的成像以最小的光毒性长时间。 2P激励时发生的两个光子由荧光团同时吸收。高浓度的所需2P激发光子的由2P激光输出的时空压缩在一个单一的焦平面实现。这限制了荧光激发的焦点，光毒性脊髓地区进一步减少焦平面之外。近红外光子也具有降低的散射，从而使成像高达约300微米的脊髓16的腹侧。 如同任何新技术，但是，2P成像移出脊髓具有必须牢记的限制。这种成像协议采用了520纳米dichroIC镜，使的绿色荧光蛋白和EYFP荧光分光分色转移绿色荧光蛋白排放到荧光通道通常保留给蓝光。由胶原在脊髓中创建第二谐波产生也出现在蓝色通道，但很容易区别于的eGFP标记的细胞，其是显著光明在此制备，并有自己的发射的一部分可识别的黄绿色通道作为胶原没有。信道混合和使用特定分光镜的发射光谱的不同部分的分离的这种技术通常是视觉或自动识别与靠近或几乎重叠的发射光谱的荧光团个人是有用的。光毒性是这个2P成像协议的另一个限制。单个成像体积内的细胞是光毒性敏感;因此，实验者应该敏锐地意识到光毒性的迹象。光毒性通常在减少或缺乏的细胞的第一明显运动，随后在漂白局部组织和/或形态变化。有几个因素可能会损坏导致光毒性或使其不适合用于活细胞成像的组织。关键是要监视灌注的RPMI-1640的激光功率和温度和氧气。此外，还必须以确保适当除去从小鼠的脊髓不会过度伸展或脊髓的压缩和不与刀片切割脊髓。如果处理不当，该外脊髓准备继续生存长达十几个小时，通过强大的细胞运动脊髓腹侧没有缩减在十小时后，抽取确定。各种组织中提取的一般拍摄的时间很长的长度，并认为可行23-26。因此，一个单一的脊髓内量化在多个区域的细胞动力学是可能的。最后，应该指出的是，细胞THA绝大多数吨未荧光标记将不会由该制备（除非天然自身荧光）进行可视化，并确认该标记的细胞与其他未标记的，表面上看不见的内源性细胞和结构元件的复杂的环境相互作用是重要的。

小鼠模型脱髓鞘，包括实验的自身免疫性脑脊髓炎（EAE）和颅内感染neuroadapted小鼠肝炎病毒（MHV），是优秀的工具来研究分子途径和与疾病相关的细胞相互作用。他们带领和支持FDA的批准有效性医药疗法，主要针对戒烟自身免疫和炎症1。然而，一旦内源性髓鞘再生失败时，目前批准的疗法不能有效地修复脱髓鞘病变在中枢神经系统。因此，修复为重点的疗法在这个阶段的疾病是慢性症状和改善生活质量的减轻是至关重要的。最近，神经前体细胞（筹备）已经走到了前列作为一个潜在的再生治疗方法针对炎症和脱髓鞘的领域。一些研究突出的NPC诱导endogeno的能力我们髓鞘再生，并直接在髓鞘2-8参与。由于筹备参与直接的髓鞘再生，就必须了解他们的动力学和相互作用移植后的内源性细胞。移植后，NPC的腹侧迁移到脑白质损伤区，然后吻侧和尾侧相对于移植部位5,9。迁移的动力学不同的应对环境线索; NPC的移植到未受损脊髓具有比的NPC移植到受损脊髓6更大的流速。后一个洄游期间，传送的NPC广泛增殖，以更高的速率在 ​​受损脊髓一个相对于一个完整的脊髓6。最后，大多数的NPC分化成少突胶质细胞，并开始直接髓鞘4,6,9。 脱髓鞘病变是复杂的，并且可以包括小区中的一个的各阶段不同的人口ctivati​​on。例如，有源多发性硬化症（MS）病变可以包括活化的T细胞，小胶质细胞M1和M1巨噬细胞，但是慢性无声的MS病变可以包括主要由具有少量炎症细胞10-13活性星形细胞的显著人口。由于效应细胞的多样性，双光子（2P）成像脱髓鞘的小鼠模型是一个非常有用的工具，以帮助了解病灶内局部细胞的相互作用。在MS和许多广泛使用的MS研究模型，大部分病灶位于脊髓，无法进入活体2P成像的区域的腹侧由于损伤深度和脊髓的高脂质含量。绕过病变内的这些问题，并研究细胞-细胞相互作用沿腹侧脊髓我们已开发出一种简单的体外 2P成像制剂6。 本研究遵循了以前的方法的出版物，这表明该过程对于移栽增强型绿色荧光蛋白（EGFP）的NPC -expressing到小鼠的脊髓以下MHV诱导脱髓鞘14 JHMV应变。五周龄小鼠感染JHMV和移植EGFP-的NPC在胸9级后第14天感染。这里介绍的协议提供了有关如何提取脊髓，作出体外琼脂糖准备，并具有增强的黄色荧光蛋白（EYFP）-expressing轴突图像移植EGFP-NPC互动的详细步骤。小鼠表达EYFP下神经元特异性大鼠Thy1启动子在此过程15被使用。只有一些轴突表达EYFP，使其可用于单个成像轴突。在这里，我们将展示7天移植后取出脊髓;然而，脊髓可以在任何时间点移植后进行萃取。虽然我们显示的NPC的相互作用受损轴突，我们的协议可以结合使用遗传荧光标记其它类型的细胞，调查了许多在整个小鼠脊髓发生细胞相互作用的。

<table border="0" cellpadding="0" cellspacing="0" width="858">
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two-photon imaging of cellular dynamics in the mouse spinal cord

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

<html>而取出的脊髓成像协议可用于可视化的脊髓内的任何荧光，我们的代表性结果证明与EYFP-轴突的eGFP-NPC相互作用。首先，我们示出了嵌入式腹侧脊髓制备图1A。接下来，我们显示了2P显微镜设置和关键部件在图1B，图2展示了EGFP和EYFP荧光在腹侧脊髓内的单个Z堆叠。收购连续的z栈可以被编译生产时间推移录像到内完整的组织分析实时细胞动力学。使用520纳米的单刃分色和一个560纳米的单刃分色光束分离器，如在协议所指出的，可以单独的eGFP和EYFP信号。各个通道可以伪彩色绿色和黄色的使用图像处理软件。 80 / 52580fig1.jpg"/&gt; 图1:脊髓和显微镜设置。 （A）脊髓中嵌入的5％琼脂糖凝胶（左）和安装在盖玻片下除去过量的琼脂糖（右）。（B）中的显微镜设置有标记的关键部件的图像。 1.水浴设置在37℃。 2.预热RPMI-1640。 3. C / L变速泵管。 4.单列直插式解决方案加热器。 5.浸渍目标。 6.数字温度计。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52580/52580fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52580/52580fig2.jpg" /> 图2:实施例2P图像腹侧脊髓内获取的未感染的，未受损的（A）的腹侧和JHMV感染，脱髓鞘（B）的脊髓的三维重建。从大鼠Thy1-EYFP小鼠移植后具有绿色荧光蛋白标记的NPC。荧光标记的轴突的伪彩色黄色和筹备的伪彩色绿色。图像分辨率:512像素;图像体积:（A）×= 239微米，Y = 259微米，Z采用26 Z-堆栈= 65微米构建间隔2.5微米分开，（B）x = 497微米，Y = 389微米，Z = 127.5微米建成使用51 Z-栈间隔2.5微米分开。

Watch this Scientific Journal Video about Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord at JoVE.com

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

成像的小鼠脊髓一种新的体外准备。该协议允许对活细胞相互作用的整个脊髓双光子成像。

细胞动力学在小鼠脊髓双光子成像

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

two-photon-imaging-of-cellular-dynamics-in-the-mouse-spinal-cord

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.2K 次观看.  University of California, Irvine. 成像的小鼠脊髓一种新的体外准备。该协议允许对活细胞相互作用的整个脊髓双光子成像。 

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

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

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

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

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

在体内脊髓的鼠标线使用双光子显微镜成像

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

科研

神经科学

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

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

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

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

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

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

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

在大鼠颈脊髓弥散成像

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

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 ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

在体内挫伤脊髓损伤后影像血管动力学的双色法

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

细胞动力学在小鼠脊髓双光子成像

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