1。大厦的脊椎稳定装置<ol><li>订购Narishige STS的紧凑型脊髓夹钳和Narishige马6N头控股适配器。 </li><li>自定义的设计和制造的不锈钢底板举行对齐Narishige的两个部分，使动物的头是支持，而其脊柱和尾巴夹紧。请记住，整个装置应适合在显微镜下的镜头，通常在降低显微镜阶段。 </li></ol> 2。动物手术<ol><li>预热空气加热装置的显微镜室，并保持在37 ° C。 </li><li>称量动物和麻醉剂混合使用100毫克氯胺酮，甲苯噻嗪15毫克和2.5毫克每公斤体重acepromazine无菌条件下，在0.9％NaCl溶液稀释。 </li><li>注射麻醉剂混合（KXA）腹腔麻醉动物。应定期脚趾捏执行，以确保深麻醉，整个实验。一半原始剂量每小时或需要补充。 </li><li>使用小动物加热垫，提供热的支持，同时执行外科手术。 </li><li>应用人工泪液对眼睛的药膏，以防止角膜脱水和损害。 </li><li>首先具有剪裁设备，然后用锋利的刀片，从切口周围地区彻底清除头发剃动物背面。 </li><li>删除剃光头发剃去皮肤表面用酒精垫消毒。 </li><li>执行一个小的（〜1.5厘米）的纵行切开皮肤所需的脊髓水平（此处显示胸椎）。 </li><li>揭露仔细缩回皮下结缔组织的背部肌肉。 </li><li>仔细分离脊柱椎旁肌，在理想的水平。执行尽可能少的切口，脱离每个棘突两侧的肌肉，然后刮分离肌肉从层表面的方式。 </li><li>选择将要删除的的椎板和脊柱两侧脊髓夹具准备进入的网站，立即延髓和尾鳍的椎板切除术。仔细取代肌肉组织，使四个夹具将被插入口袋。 </li><li>电梯与直齿尖镊子脊柱，让没有接触的脊髓弯曲的钝头剪刀椎板下安全地插入。慢慢切割一侧的骨，然后和对方进行椎板切除术。 </li><li>使用棉签或预切小明胶海绵吸收明胶海绵片插入切口限制轻微出血。使用一个小的船只cauterizer，如果有必要进一步控制出血。使用预热的无菌人工脑脊液（ACSF）冲洗组织。 </li></ol> 3。 在体内成像稳定脊柱和准备<ol><li>的Plac<html> E的动物脊椎稳定装置的底座上海拔垫和安全的头部，在头部控股适配器。 </li><li>将沿侧翼椎板切除术（图1）在事先准备好的口袋里的动物的前后轴的STS - A装置脊髓夹具。两个夹子应放置在一个角〜45 °相对于动物的rostro尾椎轴允许足够的空间，在暴露脊髓（图1）为降低水浸泡镜头。 </li><li>将第三钳在动物的身体，可以使悬浮在空气中海拔垫去除成像实验（图1）的时间后尾基础的STS - A设备。 </li><li>建立一个小Gelseal（Amersham Biosciences公司公司）在外露的脊髓以及在学联脊髓维修方便，在此解决方案中的显微镜镜头浸泡在体内成像。 </li></ol>jove_title“&gt; 4。双光子显微镜在小鼠脊髓的体内成像<ol><li>动物的脊椎稳定装置传输覆盖在显微镜内的预热室和安全上降低显微镜配售阶段的椎板切除，直镜头下的（图1）。 </li><li>下一个镜头浸水，小心地将学联的解决方案，确保它不触及脊髓夹具或Gelseal。 </li><li>使用epifluorescence找出感兴趣的领域，专注于IT。切换到激光扫描模式，并使用相应的双光子激光激发波长，dichroics和带通的荧光团的过滤器在成像组织目前在体内成像执行。 </li></ol> 5。重复成像和手术后的护理<ol><li>在成像实验结束时，请从脊椎稳定装置鼠标，并仔细擦拭周围椎板切除的面积Gelseal。以及清洁的区域。 </li><li>还原和缝合在椎板​​切除背部肌肉。 </li><li>还原和缝合皮肤在椎板切除，用优碘棉签。 </li><li>提供1毫升乳酸钠林格作为补充营养和水化溶液（百特医疗用品），以及镇痛治疗皮下注射（0.1毫克/千克丁丙诺啡）。 </li><li>管理防腐处理腹腔（每鼠0.03毫升，2.27％恩诺沙星注射抗菌解决方案）。 </li><li>广场上，直到完全恢复从麻醉中加热垫的动物和随后它单独的房子。 </li><li>每天重复防腐管理，为2-3天，手术后第3-5天，手术后镇痛治疗，每8-12小时。日常监测，以确保正常的行为，并全面复苏。 </li><li>通过相同的椎板切除术的重新成像，重新缝合的皮肤和肌肉，然后重复第2 - 4中所述的步骤。 </li><li>重新定位的先前ously使用地图作为先前所描述的10的血液血管成像区域。 </li></ol> 6。代表性的成果所有动物的程序进行体制在旧金山加利福尼亚大学的动物护理和使用委员会的指引下，按照联邦法规。的脊椎稳定装置的图片和显示设备上的显微镜镜头下的鼠标定位原理图如图1所示。允许动物的身体底下呼吸运动足够的房间，确保稳定， 在体内脊髓成像。 图2显示小胶质细胞和血管之间关系密切因为它是在体内成像在脊髓CX3CR1 GFP / +转基因小鼠18，小胶质细胞是内源性与GFP标记。 图3显示了重复的例子在体内成像，因为它是在执行相同的表达在脊髓轴突荧光蛋白（YFP - H 线 3）和小胶质细胞（CX3CR1 GFP / +小鼠）的小鼠脊髓领域。 <img alt="图1" src="/files/ftp_upload/2760/2760fig1.jpg" /> 图1。小鼠体内成像，利用双光子显微镜的脊柱的稳定。（A）定制的钢底板是用来支持和调整的STS - Narishige紧凑脊髓夹具和MA - 6N Narishige头控股适配器，如下所示（B）与脊椎稳定装置KXA麻醉麻醉的成年转基因小鼠的正确定位。插入显示立即rostrally和尾端的椎板切除术和暴露的脊髓组织脊髓夹具的位置。 &lt;IMG ALT =“图2”SRC =“/ files/ftp_upload/2760/2760fig2.jpg”/&gt; 图2在体内的高密度小胶质细胞和麻醉小鼠的脊髓血管成像。 （一）预计，但不结盟Z -叠高度密集的GFP阳性小胶质细胞（绿色）在一个CX3CR1 GFP / +鼠标在接近血管（红色标记，静脉注射罗丹明葡聚糖）的脊髓。 （二）（一）一个单一的小胶质细胞与周围的船只扩展的进程，对脊髓实质血管壁高倍率图像标记。比例尺为10μm。 <img alt="图3" src="/files/ftp_upload/2760/2760fig3.jpg" /> 图3。重复相同的轴突段和小胶质细胞，因为他们在不同的日子里搬迁重新制作映像脊髓麻醉小鼠体内成像。 （一）YFP的实验室<html> 0和5天的ELED轴突。 （二）。相同的血管结构和他们周围的小胶质细胞成像在体内的0和1 天的CX3CR1 GFP / +小鼠的脊髓。比例尺为10μm。 电影1。代表timelapse序列在体内收购从小鼠的脊髓。这个顺序显示在详细细的小胶质细胞的过程动态（绿色）和它们之间的相互作用，超过时间内密集的荧光结构填充组织的血管（红色标记，四罗丹明右旋糖酐注射）。只有纠正原始图像背景噪声，亮度，对比度和timelapse电影Z -投影顺序获得的图像栈构造，没有图像对齐，平均或个别平面的Z -选择。血管经过一个类似的范围内小胶质细胞的图像z平面。 Z平面深度：38微米。oad/2760/Davalos_Movie_1.mov“&gt;点击这里观看影片。 电影2。Timelapse序列，证明在一个单一的z平面水平的成像方法的原始稳定性。快速获取相同的单/ GFP的 CX3CR1 +脊椎稳定装置划归KXA麻醉小鼠的脊髓z平面，演示在1帧/秒的扫描速度比快的连续帧之间的最小图像位移鼠标的呼吸率。轻微的剩余位移可能是由于心跳。 <a alt="Movie 2" href="http://www.jove.com/files/ftp_upload/2760/Davalos_Movie_2.mov">点击这里观看影片。</a>

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我们什么都没有透露。

这里介绍的方法， 可以在体内成像人口稠密荧光灯在使用双光子显微镜的麻醉小鼠的脊髓细胞结构稳定和重复性。所取得的稳定，是一个特制的脊椎稳定装置和麻醉方案，减少呼吸道引起的运动伪影的结果。脊椎稳定装置允许下的鼠体呼吸的空间，可使用市售的脊髓夹具和头部安装片（图1）。高重现性好，微创的方法是，始终没有广泛的图像后处理（图和视频1），可用于实验分析产生的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>若丹明B葡聚糖</td><td> Invitrogen公司</td><td> D1841 </td><td> 70 kDa的，稀释 学联（3％W / V） </td></tr><tr><td>氯胺酮盐酸</td><td> Bionichepharma </td><td> NDC的无：67457-001-10 </td><td>注射剂，50mg/ml </td></tr><tr><td> Anased </td><td>劳埃德实验室</td><td> NADA无：139-236 </td><td>甲苯噻嗪注射剂， 20mg/ml </td></tr><tr><td> Acepromazine </td><td> Vedco </td><td> NADA无：117-531 </td><td>注射剂，10mg/ml的</td></tr><tr><td>人工泪液 软膏</td><td>凤凰 医药</td><td> NDC：57319-760 - 25 </td><td>润滑剂</td></tr><tr><td>优碘</td><td>费舍尔</td><td> 19-061617 </td><td></td></tr><tr><td>麦克弗森的韦斯科特 剪刀</td><td>世界精密 仪器</td><td> 555500S </td><td>弯曲，钝尖 剪刀</td></tr><tr><td>直钳</td><td>世界精密 仪器</td><td> 555047FT </td><td>齿尖钳</td></tr><tr><td>小血管烧灼</td><td>精细科学工具</td><td> 18000-00 </td><td></td></tr><tr><td>明胶海绵</td><td>法玛西亚，辉瑞公司</td><td>调音台轧机MM400 </td><td></td></tr><tr><td>紧凑型脊髓 夹具</td><td> Narishige </td><td> STS - 一个</td><td></td></tr><tr><td>头部控股适配器</td><td> Narishige </td><td> MA - 6N </td><td></td></tr><tr><td> Gelseal </td><td> Amersham公司 生物科学公司</td><td> 80-6421-43 </td><td></td></tr><tr><td>乳酸钠林格</td><td>百特医疗用品&lt;/ TD&gt; <td> 2B8609 </td><td></td></tr><tr><td> Buprenex </td><td> Reckit洁 制药公司</td><td> NDC编号：12496 - 6757-1 </td><td>丁丙诺啡 注射</td></tr><tr><td> Baytril </td><td>拜耳</td><td> NADA 140-913 </td><td>恩诺沙星， 抗菌注射 2.27％（20毫升） </td></tr><tr><td>加热垫 - 大</td><td>精细科学工具</td><td> 21060-10 </td><td></td></tr></tbody></table>

in vivo imaging of the mouse spinal cord using two-photon microscopy

在体内成像已遗传工程小鼠表达荧光蛋白在特定的细胞类型 2-3显著扩大我们的生理和病理过程知识在众多的组织在 体内 4-7使用双光子显微镜 1 。在中枢神经系统（CNS）的研究，已被广泛的应用在体内成像在大脑中，这已经产生了众多新颖的细胞，如神经元，星形胶质细胞，小胶质细胞，在行为往往是意外的结果生理或病理条件下 8-17 。然而，大多是复杂的技术问题，有限的生活小鼠脊髓的研究在体内成像的实施。特别是，对脊髓的解剖毗邻的肺部和心脏产生显著运动伪影，使成像的生活脊髓一项艰巨的任务。 </P&gt; 我们开发出一种新的方法，克服了稳定脊柱，减少呼吸道引起的运动，从而促进利用双光子显微镜的图像在体内小鼠脊髓的脊髓造影固有的局限性。这是通过深麻醉方法相结合的一个定制的脊柱稳定装置，导致呼吸道引起的运动的一个显着减少。这个视频协议显示了如何公开一个可以维持稳定的生理条件下过长时间保持组织损伤和出血最低生活脊髓的小面积。具有代表性的原始图像在高分辨率收购体内详细的小胶质细胞和血管之间的密切关系。一个timelapse序列显示了在生活小鼠脊髓小胶质细胞过程的动态行为。此外，一个相同的Z -帧的连续扫描显示S的出色的稳定性，这种方法可以实现图像和/或timelapse电影，​​不需要图像对齐收购后产生的栈。最后，我们表明这种方法可用于如何重新审视和重新映像在稍后的时间点脊髓同一地区，从而在体内持续的生理或病理过程的纵向研究。

Watch this Scientific Journal Video about In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy at JoVE.com

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

微创的协议，以稳定脊柱鼠标和执行重复<em&gt;在体内</em&gt;脊髓成像，利用双光子显微镜描述。这种方法结合了脊柱的稳定装置和麻醉方案，以尽量减少引起的呼吸运动和生产没有对齐或其他处理后的原始图像数据，需要。

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 ...

in-vivo-imaging-of-the-mouse-spinal-cord-using-two-photon-microscopy

Research

JoVE Journal

Neuroscience

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

23.7K 次观看.  University of California, San Francisco. 微创的协议，以稳定脊柱鼠标和执行重复在体内脊髓成像，利用双光子显微镜描述。这种方法结合了脊柱的稳定装置和麻醉方案，以尽量减少引起的呼吸运动和生产没有对齐或其他处理后的原始图像数据，需要。

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

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 ...

科研

神经科学

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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 ...

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.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

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 ...

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 ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...