我们感谢Bakhos A. Tannous，博士为我们提供的rAAV-EGFP载体，载体的开发和生产在马萨诸塞州的查尔斯顿，马萨诸塞州总医院，神经科学中心的主任，约翰·王海提供技术援助。这项工作是支持的补助R01 NS050408（JS）由美国国家神经疾病与中风研究所。

描述的外科手术已经批准了哥伦比亚大学实验动物护理和使用委员会（IACUC）。 1。设备和病毒颗粒悬浮液的制备<ol><li>清洁和消毒的设备，消毒的外科手术器械和V形缺口，将用于修复椎骨L1尖峰。 </li><li>拉和锥玻璃吸管。我们使用的移液管的前端的直径为40μm，并在以20°的角度倾斜的。消毒的玻璃吸管。 </li><li>设置的立体定位框架，安装到操纵器上的微量喷射器，喷射器连接到控制器。 </li><li>微量使用压缩装配套件安装的玻璃吸管。 </li><li>将柱塞微量矿物油填补了注射器。油红O（1 - （2,5 - 二甲基-4-（2,5-dimethylphenylazo）苯基偶氮）-2 - 萘酚）可被加入到矿物油，以增加它的visibility。重新将活塞推给小费的方式。小心避免气泡。 </li><li>准备病毒颗粒悬浮在生物安全柜。在冰上解冻冷冻病毒，只是在使用前，用无菌磷酸盐缓冲盐水所需的颗粒浓度稀释。 </li><li>将微量注射器持有人。 </li><li>吸取5μl病毒悬液到一个小塑料片， 如封口膜。降低玻璃吸液管头下降到拉活塞，填补了微量。创建一个小气泡，在移液管尖，以防止它从堵塞。 </li></ol> 2。椎板切除术<ol><li>准备手术部位用消毒液擦拭工作台和加热垫。 </li><li>麻醉鼠标。我们使用吸入异氟醚麻醉诱导过程中，在维修过程中的2％-3％）（3％。 </li><li>将每只眼睛以保护眼睛免受干燥过程中的润滑剂操作。 </li><li>剃须从下背部的鼠标的颈部和皮草消毒皮肤与交替的局部消毒剂如洗必泰或聚维酮碘和70％乙醇的湿巾。隔离无菌制备的网站与手术被单和渗透布比卡因（0.25％，生理盐水稀释1:10）的切口部位。 </li><li>在尾端的肋骨沿中线（2-3厘米），切开皮肤，分离筋膜覆盖脊柱。 </li><li>由于脊髓停止生长早期发育过程中的比脊柱，脊髓节段L4位于下方的第一腰椎（L1）。 L1椎体位于骶管的脊椎骨持有的最后一对肋骨。识别和消除小脊髓肌肉和韧带连接到其背表面暴露椎体L1。 </li><li>轻轻抬起，保持脊椎的L1与一个ADSON的钳。使用专用的椎板钳，删除背portioN的椎体（脊椎和叶片）和暴露脊髓。避免损伤脊髓。 </li><li>转移鼠标在立体定位框架到加热板。随后的操作过程中，监测温度的鼠标。 </li></ol> 3。注射<ol><li>修复椎体L1与V型缺口的峰值。尖峰必须稳定，使脊柱的椎体呼吸过程中不动。 </li><li>带来的微量近点儿，让吸头以上椎板网站。降低活塞，直到你看到病毒悬液退出吸液管。用无菌棉棒取出液滴。 </li><li>移液管尖头定位在最延髓部的暴露的脊髓。中心后正中沟以上的吸管，然后将尖端500μm的横向。降低头电源线和刺破硬脑膜表面，如果您使用的是未斜切的玻璃吸管，用一个斜面钢套管穿刺的d市区重建局。放下的前端的玻璃吸移管300微米到脊髓。 </li><li>注射1微升病毒悬浮液，在200升/分钟的速率。 </li><li>在注射完毕，等待至少2分钟，才慢慢地缩回吸管。 </li><li>重复步骤3.3至3.5，最的尾部的露出的脊髓的病毒载体在脊髓节段L4实现完全分布。两个注射部位位于延髓和尾侧的L4段，以避免在目标区域中的组织损伤的。 </li></ol> 4。伤口愈合<ol><li>发布椎体L1的V型缺口夹，删除鼠标的立体框架。 </li><li>缝合筋膜与5.0薇乔。封闭的筋膜椎板网站提供保障。 </li><li>关闭肌肤的尼龙手术缝合或钉书钉。 </li></ol> 5。术后护理<ol><li>鼠标传输到恢复笼被褥与柔软，nonparticular的。将它放在吨他一边舒适的呼吸。监控动物，直到它是完全清醒的，能行走，并开始饮用。 </li><li>我们提供术后镇痛的72小时：每日皮下注射的卡洛芬（5.0毫克/公斤）。 </li><li>监测术后第3天恢复日常检查，然后隔日或每周3天，直到试验完成。 </li><li>去除皮肤缝合或钉书钉，手术后7〜10天，当伤口完全愈合。 </li><li>实验完成后，安乐死动物。 </li></ol>

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作者宣称，他们没有竞争的金融利益。

立体定向的载体注射可针对脊髓神经元的应用程序，如神经网络映射的基础上跨突触传播的病毒6,7或optogenetic解剖，轴突引导再生过程中从伤病中9,10，或基因治疗用于预防或治疗神经退行性疾病11， 12。病毒载体用于基因操作的脊髓研究躯体感觉，运动和植物神经途径9,10,13-15。鼠标是最广泛使用的模式生物的研究涉及立体病毒载体注射到大脑或脊髓，但该技术已?...

有条件的基因操作在小鼠的先进技术，使多方面的探索中枢神经系统的的突触途径和功能连接方法。转基因可能是受小分子效应如强力霉素，四环素控制的反式激活，它可以被设计为充当一个阻遏或激活基因转录，或他莫昔芬承认的雌激素受体​​1的配体结合域的突变作用于。通常是不可逆的转基因修饰的实现由脱氧核糖核酸（DNA）的重组酶。华创（的原因重组）和FLP（flippase重组酶）催化loxP位（轨迹，穿越x，P1）或首次登记税（flippase识别目标）的网站，分别为1的DNA片段，这些片段两侧切除，反转或易位。应用范围包括基因的激活或沉默和诱导型核糖核酸（RNA）干扰<sup&gt; 2。可以使用的条件表达的荧光或酶如β-半乳糖苷酶或碱性磷酸酶的记者标记神经元，并检查它们的局部组织和连接3。大型诱变项目在北美（ <a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a> ）和欧洲（ <a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm</a> ）的小鼠胚胎干细胞克隆库有条件的靶基因和陷阱，最终将覆盖整个小鼠基因组中。鼠标线膨胀的数目，反映根据特定的神经元的选择性基因操作（ <a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index</a>某一特定人群的启动子或基因座的DNA的重组酶，可以划线，从这些克隆产生的小鼠<a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">PHP</a> ）。 <p class=“jove_content”&gt;然而，限制基因操作不同人群的神经元或感兴趣的特定区域可能不实现通过基因单独定位，如果还没有已知的或不表达在该区域的所有神经元到神经元特异性的启动子人口的利益的兴趣。在脊髓中，实验设计，可能需要一个或两个头尾段空间限制的基因操作。立体定位注射病毒载体表达Cre或FLP，允许限制基因重组小鼠脊髓中的DNA片段由loxP位或frt位点两侧的区域，即所谓的两侧装接loxP的或flrted等位基因。与组成DNA重排，这将导致从杂交育种与重组表达小鼠的动物，这种策略还提供了时间控制基因的激活或沉默。病毒载体编码两侧装接loxP或调情转基因提供了一个逆向选择的基因操作在小鼠体内表达的corresponding重组的神经元特异性启动子的下游。重组表达载体亲和力的神经元是4。高容量（无胆）腺病毒，腺相关病毒，单纯疱疹病毒和慢病毒常​​用嗜神经载体。选择合适的病毒用于研究的问题是实验设计的一个重要组成部分。的转基因的大小，送货路线，特异性感染的神经元，而不是神经胶质细胞，感染的疗效，炎性和毒性副作用需要考虑4。 在这里，我们描述了立体的病毒载体注射到脊髓背角，采用的技术，我们的条件在我们的研究对疼痛的神经生物学基因调控。背角接收传入的初级躯体感觉神经元的输入，包括伤害性神经元。当地的interneurons处理信息，然后投射神经元的传递从背角的大脑5。我们展示了一种嗜神经的重组腺相关病毒（rAAV），组成性激活的巨细胞病毒启动子表达增强型绿色荧光蛋白（EGFP）下背角神经元在脊髓节段L4感染。

<table border="1"><tbody><tr><td> 材料名称 </td><td> 公司 </td><td> 目录编号 </td><td></td></tr><tr><td>脊髓底板</td><td>大卫KOPF工具</td><td> 912 </td><td></td></tr><tr><td>小动物立体定位仪</td><td>大卫KOPF工具</td><td> 900 </td><td></td></tr><tr><td>鼠标气体麻醉头支架</td><td>大卫KOPF工具</td><td> 923-B </td><td></td></tr><tr><td>可调底座支架</td><td>大卫KOPF工具</td><td> 982 </td><td></td></tr><tr><td> V型缺口尖峰</td><td>大卫KOPF工具</td><td> 987 </td><td></td></tr><tr><td>小动物的温度控制系统</td><td>大卫KOPF工具</td><td> TCAT-2LV </td><td></td></tr><tr><td> ADSON钳</td><td>精细科学工具</td><td> 11006-12 </td><td></td></tr><tr><td>椎板切除术钳</td><td>精细科学工具</td><td> 11223-20 </td><td></td></tr><tr><td> UltraMicroPump（一）SYS-MICRO4控制器</td><td>世界精密仪器</td><td> UMP3-1 </td><td></td></tr><tr><td>微量，65RN </td><td>汉密尔顿</td><td> 7633-01 </td><td></td></tr><tr><td> RN压缩接头，1毫米</td><td>汉密尔顿</td><td> 55750-01 </td><td></td></tr><tr><td>高硼硅玻璃毛细管</td><td>世界精密仪器</td><td> 1B100F-4 </td><td></td></tr><tr><td> Microgrinder </td><td>成重</td><td> EG-44 </td><td></td></tr></tbody></table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

实质内注射病毒载体使在不同人群的中枢神经系统的神经元或特定区域的条件的基因操作。我们展示了一个立体的注射技术，可以使靶基因的表达或沉默的小鼠脊髓背角。手术是短暂的。它需要一个单一的椎椎板切除术，提供快速恢复脊柱的动物和不受影响的活力。在低速和使用微量的斜角玻璃套管控喷射的小矢量悬浮液体积减少组织损伤。局部免疫反应的矢量取决于采用的病毒的内在特性;根据我们的经验，这是轻微和短暂的，使用时的重组腺相关病毒。如增强型绿色荧光蛋白报告基因的方便的载体，空间分布监测和疗效和蜂窝指定的转染ficity。

成功转染产生强大的基因的表达，不惜背角对侧，腹角和背根神经节神经元的注入背角（ 图1）。 <img src="/files/ftp_upload/50313/50313fig1.jpg" alt="图1" /> 图1。 （A）表达的荧光记者左背角L4脊髓，两个星期后的rAAV-EGFP的立体定向注射（血清型AAV2 / 8，9个基因组拷贝/ EGFP（绿色） 转染背角神经元。微升）。神经元进行免疫染色的神经?...

Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

病毒载体可以进行有针对性的基因操作。我们提出了一个条件性基因的表达或消融方法在小鼠脊髓，采用立体的病毒载体注射到脊髓背角，初级躯体感觉传入中枢神经系统的神经元之间的突触接触的一个突出的部位。

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

stereotaxic-injection-viral-vector-for-conditional-gene-manipulation

Research

JoVE Journal

Neuroscience

30.1K 次观看.  Centre National de la Recherche Scientifique (CNRS). 病毒载体可以进行有针对性的基因操作。我们提出了一个条件性基因的表达或消融方法在小鼠脊髓，采用立体的病毒载体注射到脊髓背角，初级躯体感觉传入中枢神经系统的神经元之间的突触接触的一个突出的部位。

视频: Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

科研

神经科学

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

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 ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

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 ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

直接注射慢病毒载体突出大鼠脊髓中的多种运动途径

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...