这项工作得到了科学和技术部的支持, MOST104-2311-B-010-010-MY3、MOST106-2321-B-010-012、国家卫生研究所赠款 NHRI-EX106-10429NI 和特色领域研究中心方案赠款来自教育部通过台湾国立阳明大学脑研究中心和博士后奖学金授予 MOST106-2811-B-010-031 (Y.C.)、MOST105-2811-B-010-036 和 MOST106-2811-B-010-030 (H 张佑启)。

这里所描述的动物协议已经得到国立阳明大学动物保育委员会的批准。
1. 在立体定向装置中制备新生儿幼崽的支架
<ol><li>制作头托盘: 将1.5 毫升离心管 (15 毫米长) 的底部切割成适合新生儿幼崽头部的形状, 并除去1/5 的管壁。</li>
	<li>取一个合适大小的吸管尖盒, 适合于立体定向设备的底座, 并取下顶盖。将步骤1.1 中的头托盘和组织嵌入盒贴在热熔胶粘剂的刀尖托盘底座上。组织嵌入盒的高度为7毫米, 用于支撑幼犬的颈部和身体在头部固定位置。</li>
	<li>将整个设置放置在标准的立体定向设备中。</li>
</ol>2. 30G 注射用不锈钢针的制备
<ol><li>用氯仿浸泡3天前清洁30G 注射器针。</li>
	<li>使用镊子小心, 慢慢地从它的聚丙烯轮毂在一个化学罩拔出针。</li>
	<li>用绝对乙醇清洗针头20分钟后, 70% 乙醇漂洗 3 x 10 分钟在一个振动筛在 50 rpm。让针头风干, 并将清洗过的针头储存在室温 (RT) 的清洁箱中, 直到使用。</li>
</ol>3. 准备用于微注射油管的适配器
<ol><li>用 PE10 聚乙烯管 (PE10 管) 将微升注射器连接到30G 注射针。准备一 PE20 聚乙烯油管 (PE20 管) 适配器, 用于连接注射针和微升注射器。使用适配器是必要的, 因为 PE10 管的直径比26G 微升注射器的针头小得多。</li>
	<li>准备微升注射器的油管: 用5厘米的 PE20 管连接预清洗的30G 注射针, 并用即时胶水密封接头。这种油管适配器是可重用的。 注: 如果注射注射器的针头为 30G, 则不需要此适配器的准备 (步骤 3.1) 和使用 (步骤 3.3)。</li>
	<li>用微升注射器 (10 µL) 将油管适配器 (在步骤3.1 中制备) 连接起来。</li>
	<li>将 PE10 管的一端连接在 PE20 油管适配器的30G 针上 (不超过60厘米)。将新的30G 注射针安装到 PE10 管的另一端。</li>
</ol>4. 用蒸压蒸馏水、染料和病毒装载微注射管
<ol><li>取出微升注射器的柱塞。使用25G 注射器加载微升注射器及其连接的 PE 管与蒸压蒸馏水, 以消除空气从油管。</li>
	<li>将柱塞放回微升注射器, 并推入柱塞直到2µL 的蒸馏水留在桶中。不要让蒸馏水的体积低于2µL。</li>
	<li>小心地将微升注射器安装在微流速注射器泵上。</li>
	<li>吸管少量0.1% 快速绿色染料 (准备在0.9% 盐水和过滤与0.22 µm 过滤器) 和病毒液体到一块 parafilm。</li>
	<li>提取少量空气, 使气泡在30G 注射针和 PE10 管之间的连接处可见, 然后将0.7 µL 过滤后的快速绿色装入管内, 以测试微注射油管中的流体流动。</li>
	<li>取出另一小部分空气, 使第二个气泡接着将病毒液体装入微注射油管。</li>
	<li>将30G 显微注射针固定在立体定向装置的手臂上。</li>
</ol>5. 低温对新生小鼠的麻醉作用
<ol><li>把小狗放在乳胶手套袖子里, 把它浸泡在被粉碎的冰上, 脖子上5分钟。</li>
	<li>用镊子捏住幼崽的脚, 以确保它的脚没有退缩反应。</li>
	<li>将小狗与乳胶手套套放在头托盘, 并在乳胶套周围放置一些粉碎冰, 以保持它冷低温麻醉。</li>
	<li>在小狗眼中应用兽医软膏, 以防麻醉时的干燥, 如果可能的话。</li>
</ol>6. 显微注射
<ol><li>用70% 乙醇彻底擦拭立体定向仪, 制备无菌手术。将手术器械浸泡在70% 乙醇中消毒。</li>
	<li>用70% 乙醇擦洗幼崽的头部。找到头骨上的地标 lambda 并用标记笔标记 lambda。将针尖对准 lambda, 将前后 (AP) 和内侧侧 (ML) 坐标设置为零。</li>
	<li>根据目标站点的 X 和 Y 坐标将注射臂移动到目标站点。对于产后日 (P) 2 只幼崽的纹状体, 其座标为: AP, +2.4 毫米前的 lambda;ML, ±1.0 毫米侧向中线;背腹 (DV), −1.7 毫米从头骨。用钢笔标记 PE10 管内快速绿色染料的位置。</li>
	<li>把30G 注射针慢慢地穿透皮肤和头骨, 然后把针尖拉起来, 直到它在头骨的表面停止。将 DV 坐标设置为零。</li>
	<li>慢慢降低30G 注射针, 直到到达目标站点的 DV 坐标。等待1分钟, 让实质恢复其正常形状。运行微注射程序 (100 nL/分钟)。</li>
	<li>确保快速绿色染料的标记在 PE 管中移动, 以确保病毒液体注入大脑。</li>
	<li>在微注射结束后等待1分钟, 然后慢慢地将针从1/2 高的 DV 深度上升到三十年代以内。三十年代以后, 慢慢地从幼崽的头上取出针头。</li>
	<li>重复步骤6.2 至 6.7, 直到所有目标站点的 microinjections 完成。</li>
</ol>7. 幼犬术后恢复
<ol><li>在33摄氏度孵化器中热身20分钟的幼崽。检查幼犬从低温麻醉中恢复每5分钟, 直到幼犬恢复足够的意识, 以维持胸骨卧床。</li>
	<li>幼崽完全恢复后, 把幼崽送回大坝。</li>
</ol>

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作者没有什么可透露的。

在本研究中, 我们展示了一种简单可靠的立体定向方法, 将 AAV 病毒注入新生小鼠脑纹状体。在 P2, 我们将 AAV eGFP 病毒杏仁 Ai14 小鼠纹状体中, 然后分析了 P14 的报告基因表达。我们发现 AAV 转基因 GFP 阳性细胞在整个纹状体 rostrocaudal 水平。此外, 几乎所有的 GFP 阳性细胞都在巴细胞中共同表达了 tdTomato 报告基因, 这表明 loxP DNA 重组是由 AAV 介导的。我们进一步证实了这种方法的有效性, 通过 microinjections AAV eGFP 的纹状体Foxp2佛罗里达州/佛罗里达州小鼠 P2。双重标记表明, 许多自动增值服务转基因 GFP 阳性细胞缺乏 Foxp2 免疫反应在 P9 纹状体, 表明成功的条件删除Foxp2基因的新生儿纹状体。杏仁自动增值服务的体积与受感染脑区的大小相关。随着注入量的 200 nL, gfp 阳性细胞广泛发现在整个新生儿纹状体, 和一些分散的 gfp 阳性细胞也存在于周围的皮层纹状体。随着注入量的 50 nL, GFP 阳性细胞局部被限制在新生儿纹状体。因此, 滴定注射自动增值服务的体积对脑区神经元种群的最佳传导具有重要意义。此外, 转导率也取决于病毒制剂的效价。可以想象, 使用不同启动子的转基因小鼠可实现精确靶向特定细胞类型。
有几个外科技巧成功注射自动增值服务到感兴趣的大脑区域。首先, 由于小鼠幼崽的大小和在手术中用手固定鼠标头位置的困难,10的新生小鼠很难进行立体定向脑手术。虽然先前的研究表明新生儿立体定向手术, 但它需要两个人进行显微注射在手术期间11。我们克服了这些问题, 开发了一个定制的头部固定装置, 可以在脑外科手术中牢固地保护幼头。整个过程可以由一个人来执行。新生小鼠的脑大小因不同的遗传背景、产妇护理和幼崽的年龄而变化。我们自制的设备的优点是, 它允许一个温和但固定的新生鼠头, 这是执行高精度立体定向脑外科手术的关键。其次, 由于软皮肤的弹性和颅骨的薄壁, 在新生幼崽中, 头部的颅骨钻削后的皮肤切口很难进行。为了克服这个问题, 有必要在将注射针降低到目标脑区之前, 用针尖刺穿皮肤和头骨。第三, 检查 PE 管内可见染料的流动, 确保注射过程中微量注射的进展。如果染料不在 PE 管中移动, 应努力解决问题, 包括油管内的流体可能泄漏和堵塞的针头。请注意, 此方法适用于在 P5 前通过皮肤可见的新生幼崽。对于年龄超过 P5 的幼崽, 它可能需要在皮肤上切开以暴露 lambda。注意, 光照到 P4 和 P5 的鼠标头可能有助于找到地标 lambda。虽然我们还没有在新生大鼠中进行立体定向手术, 但我们认为该技术适用于大鼠幼崽的某些修饰。
携带基因表达的病毒的发病和最大表达在注射入大脑14、15后需要数天或几周。在本研究中, 我们发现 AAV eGFP 病毒介导的 loxP DNA 重组的新生儿巴神经元发生早在8天后病毒传导。前一项研究报告说, AAV 介导的在新生鼠幼崽的嗅球中可以检测到3天后, 自动增值服务15的散装注射。可以想象, 早期的 AAV 介导的基因活动是一个优势, 研究发展事件, 不断发生在产后大脑。
总之, 我们开发的新生儿立体定向技术不仅用于有条件地删除特定脑区的基因, 而且还可以向新生小鼠的特定脑区注入药理试剂和神经元示踪剂。虽然我们只做了 microinjections 的新生儿纹状体在本研究中, 原则上, 我们的立体定向新生儿脑外科可以应用于特定的大脑区域, 如果特定区域的坐标, 新生儿大脑可用。考虑到不同阶段的不同遗传背景的新生小鼠大脑大小的变异性, 研究人员必须通过经验主义地确定靶点的精确坐标。最后, 通过立体定向 microinjections 自动增值服务表达基因修饰 optogenetic3和 chemogenetic 蛋白4和神经元活动指标5进入特定脑区, 可以探讨神经元的作用。活动对脑后发育的大脑在细胞类型和电路特定水平的分辨率。

现代对大脑结构和功能的研究通常需要神经元细胞中特定基因的基因调控。为了探讨不同基因的功能, 转基因小鼠携带突变等位基因, 包括敲除和敲入的基因, 已被常规生成。成人啮齿动物立体定向脑外科是一种标准的方法, 当地提供药物, 病毒, 示踪剂和其他试剂到特定区域的啮齿动物大脑1,2。将立体定向脑外科应用于转基因小鼠, 允许基因调控小鼠大脑特定神经元群中的基因功能和神经元活动。细胞类型特定的操作提供了一个强大的方法来破译神经元功能的复杂神经回路的大脑3,4,5。
神经系统的神经发育始于早期胚胎阶段, 发育过程在出生后持续到幼年期。神经系统的产后成熟包括神经回路的精确突触布线, 这对于大脑6的生理和认知功能是必不可少的。因此, 研究新生儿时间窗发生的发育事件, 不仅对了解正常的神经发育有重要意义, 而且还可以为神经发育和精神疾病的发病机制提供深入的认识7 ,8。虽然成人啮齿目动物的立体定向脑外科手术方法29, 但在互联网上有少量的协议可供新生小鼠10,11的立体定向脑外科手术使用。事实上, 在新生小鼠幼崽的大脑中对试剂进行立体定向 microinjections 是困难的, 因为新生儿幼犬的头部太脆弱, 无法固定在标准的立体定向设备中。然而, 在转基因小鼠中应用立体定向脑手术是可行的12。在这里, 我们描述了一个简单的方法, 自制的设置, 进行立体定向脑外科手术的新生鼠幼崽。我们证明, 这种技术允许一个有条件地删除 floxed 基因 microinjecting AAV 表达的 DNA recombinase 到报告基因小鼠纹状体和有条件 floxed 转基因小鼠。该技术也适用于向野生型小鼠的新生纹状体提供试剂。

<table><tbody><tr><td>30G PrecisionGlide Needle</td><td>Becton Dickinson</td><td>REF 305106</td><td></td></tr><tr><td>Chloroform</td><td>JT Baker</td><td>9180-03</td><td></td></tr><tr><td>Hamilton MICROLITER Syringe</td><td>Hamilton&amp;nbsp;</td><td>80300</td><td>30G needle fit for PE10 tube; 26G needle needs a PE20 adaptor</td></tr><tr><td>Polyethylene tubing PE20</td><td>Becton Dickinson</td><td>427406</td><td></td></tr><tr><td>Polyethylene tubing PE10</td><td>Becton Dickinson</td><td>427401</td><td></td></tr><tr><td>Micro Flow Rate Syringe Pump</td><td>Longer Precision Pump Co.</td><td>TJ-2A (Controller) and L0107-2A (Drive Unit)</td><td></td></tr><tr><td>25G syringe</td><td>Becton Dickinson</td><td>REF 302105</td><td></td></tr><tr><td>Fast green</td><td>Sigma-Aldrich</td><td>F-7252</td><td>0.1%</td></tr><tr><td>Standard Stereotaxic Instruments</td><td>RWD Life Science</td><td>68037</td><td>Without using 68030 Mouse/Neonatal Rat Adaptor</td></tr><tr><td>Anti-FOXP2 antibody</td><td>Abcam</td><td>ab16046</td><td>Rabbit polyclonal to FOXP2, 1:4,000</td></tr><tr><td>Anti-RFP antibody</td><td>Abcam</td><td>ab65856</td><td>Mouse monoclonal to RFP, 1:1,000</td></tr><tr><td>BX63 microscope</td><td>Olympus</td><td>BX63</td><td></td></tr><tr><td>LSM 880 confocal microscope</td><td>Zeiss</td><td>LSM 880</td><td></td></tr><tr><td>Goat anti-rabbit conjugated Alexa fluor594</td><td>Jackson lmmunoReserch Laboratories Inc.</td><td>111-585-003</td><td></td></tr><tr><td>AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40</td><td>Penn Vector Core</td><td>AV-9-PV1848</td><td>Lot # CS0987, 5.506x10&lt;sup&gt;13&lt;/sup&gt; (GC/mL)</td></tr><tr><td>AAV9.chicken actin-eGFP</td><td>AAV core, Institute of Biomedical Sciences, Academia Sinica, Taiwan</td><td>N/A</td><td>1x10&lt;sup&gt;14&lt;/sup&gt; (GC/ml)</td></tr><tr><td>B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td><td>The Jackson Labtorary&amp;nbsp;</td><td>007914</td><td>Ai14</td></tr><tr><td>B6(Cg)-Foxp2tm1.1Sfis/CfreJ</td><td>The Jackson Labtorary&amp;nbsp;</td><td>026259</td><td>Foxp2&lt;sup&gt;fl/fl&lt;/sup&gt;</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline</td><td>Corning&amp;nbsp;cellgro</td><td>21-030-CVR</td><td></td></tr></tbody></table>

stereotaxic surgery for genetic manipulation in striatal cells of neonatal mouse brains

许多基因是在胚胎大脑中表达的, 其中有些是在出生后大脑中不断表达的。对于这种持续表达的基因, 它们可以调节新生儿大脑的发育过程和/或生理功能。为了研究大脑中特定基因的神经生物学功能, 在脑中灭活基因是必不可少的。在这里, 我们描述了一个简单的立体定向的方法, 以灭活基因表达在转基因小鼠在新生儿时间窗口。AAV eGFP 病毒通过立体定向脑外科术, 杏仁于产后 (P) 2 Ai14 报告基因小鼠纹状体。在 P14 纹状体中检测到 tdTomato 报告基因表达, 提示 AAV 转基因细胞中成功的 loxP 介导的 DNA 重组。我们进一步验证了这一技术, 通过 microinjecting AAV eGFP 病毒的 P2Foxp2佛罗里达州/佛罗里达州小鼠。荧光蛋白和 Foxp2 的双重标记表明, gfp 阳性细胞在 P9 纹状体中缺乏 Foxp2 免疫反应, 提示 AAV eGFP 细胞 Foxp2 蛋白质的丢失。这些结果表明, 在 floxed 转基因小鼠的新生儿脑中, stereotaxically 杏仁 AAV eGFP 病毒对特定神经元群有有效的遗传缺失。总之, 我们的立体定向技术为新生小鼠大脑中的基因操作提供了一个简单的平台。该技术不仅可以用于删除新生儿大脑特定区域的基因, 而且还可用于注射药理药物、神经元示踪剂、基因修饰光遗传学和 chemogenetics 蛋白、神经元活动指标和其他试剂进入新生鼠脑纹状体。

在第一组实验中, 我们杏仁了 200 AAV9 的 hSynapsin. eGFP WPRE SV40 病毒 (AAV eGFP, 1/10 Dulbecco 磷酸盐缓冲盐水稀释), 表达了与 GFP 的 DNA recombinase 与绿色荧光蛋白融合为 P2 小鼠的 Ai14 纹状体。Ai14 小鼠在 tdTomato 的 loxP 侧 (floxed) 停止盒 (图 2F) 的基础上, 表达了对该基因的报道。大脑是在 P14 染色的 GFP 和 tdTomato 中收获的。许多 AAV 转基因 GFP 阳性细胞存在于整个纹状体 (图 2A, C), 表明广泛感染的巴细胞 AAV-eGFP 病毒。类似的广泛表达 tdTomato 也发现在纹状体 (图 2B, C)。经显微检查, 在高倍率, 我们发现几乎所有 GFP 阳性巴细胞共同表达的 tdTomato 报告基因 (图 2A'-C ')。此外, 在苍白 (图 2D) 和黑质柑橘 (图 2E)、striatonigral 和 striatopallidal 投影神经元的目标区域中, 在推测的轴突终端中检测到 tdTomato 信号。13. 这些结果表明成功的 loxP 介导的 DNA 重组是由 AAV 介导的新生儿巴神经元中的疫苗活性表达引起的。
在第二组实验中, 我们将50杏仁的 AAV eGFP 病毒转化为Foxp2fl/fl 小鼠的纹状体, 在 loxP (图 3A-C, F) 上携带 Foxp2 侧的等位基因, 并在 P2 捕获大脑. 无 Foxp2+;gfp+双标记细胞在纹状体中发现, Foxp2 蛋白在 GFP 阳性细胞中缺失 (图 3A'-C ')。在控制实验中, Foxp2+;GFP+双标记细胞存在于Foxp2 AAV-eGFP 控制病毒 (图 3D) 的纹状体和异型Foxp2fl 小鼠纹状体 AAV-eGFP病毒 (图 3E)。结果表明, Foxp2基因在新生儿纹状体 AAV eGFP 转基因细胞中被明确删除。
综合AAV-eGFP 的结果;Ai14和AAV-eGFP;Foxp2 的小鼠, 我们开发的脑立体定向手术技术, 适于在新生小鼠大脑特定区域有条件地删除基因。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57270/57270fig1.jpg"> 图1。新生小鼠的立体定位仪和自制头固定支架。(A)立体定向注射系统由以下设备组成: i. 注射器泵 (控制器);二、注射器泵 (驱动单元);iii. 微升注射器 (10 µL);四、PE20 油管适配器;五、PE10 管;六、30G 注射针;七、立体定向装置;八. 吸管提示盒转换的新生儿幼崽平台;西: 冰被压碎了。(B)组织嵌入盒的高度约为7毫米。1.5 毫升离心管的按钮长约15毫米。(C)鼠标头是固定在自制的头固持有人。箭头表示新生小鼠头部的标志性 lambda。<a href="https://cloudflare.jove.com/files/ftp_upload/57270/57270fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57270/57270fig2.jpg"> 图2。P14 Ai14 小鼠纹状体中 tdTomato 在 AAV eGFP DNA 重组后的表达.AAV eGFP 病毒被 sterotaxically 注射到 Ai14 小鼠 P2 纹状体中。P14 对大脑进行了分析。(A)在纹状体 (Str) 中存在许多 GFP 阳性细胞。(B)纹状体中也存在许多 tdTomato 阳性细胞。(C)合并图像显示了荧光蛋白和 tdTomato 在纹状体中的广泛联合定位。c 中盒装区域的共焦图像分别以 "-c" 的高放大倍数显示。所有 GFP 阳性细胞 (绿色) 共同表达 tdTomato (红色) 在纹状体。(D、E)在巴投射神经元的靶区内检测到 tdTomato 信号, 包括球苍白 (GP;D) 和黑质柑橘 (信噪比;E). (F) AAV eGFP 和 Ai14 转基因结构的示意图。Ctx: 皮质;9月: 隔膜;Hipp: 海马;MB: 中脑。刻度条在 a (C), 200 µm;a ' (为 '-C '), 10 µm;d (d 和 E), 100 µm.<a href="https://cloudflare.jove.com/files/ftp_upload/57270/57270fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57270/57270fig3.jpg"> 图3。AAV eGFP 病毒 intrastriatal injecions 对新生儿纹状体中Foxp2基因的有条件删除.Intrastriatal 注射 AAV eGFP 病毒是在 P2 Foxp2佛罗里达州/佛罗里达州 (C ') 和Foxp2fl(E) 小鼠进行的。P9 对大脑进行了分析。(丙)GFP 阳性细胞 (A, c, 绿色) 缺乏 Foxp2 免疫反应 (B, c, 红色) 的纹状体的Foxp2fl/佛罗里达州小鼠与 AAV eGFP 病毒。c 中的盒装区域分别以 "-c" 的高放大倍数显示。(D)巴细胞共表达 GFP 和 Foxp2 是存在的纹状体, 注射 AAV eGFP 控制病毒。(E) GFP 阳性细胞共表达 Foxp2 (箭头) 存在于Foxp2 鼠的纹状体中, AAV eGFP 病毒。(F) AAV eGFP 病毒和Foxp2 的转基因小鼠的示意图。刻度条在 a (C), 200 µm;a ' (为 '-C ', D 和 E), 20 µm.<a href="https://cloudflare.jove.com/files/ftp_upload/57270/57270fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains at JoVE.com

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

新生小鼠脑巴细胞基因调控的立体定向手术

我们描述了一个立体定向手术的协议, 一个自制的头固定装置 microinjecting 试剂进入新生鼠脑纹状体。这种技术允许在新生小鼠大脑特定区域的神经细胞中进行基因操作。

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

stereotaxic-surgery-for-genetic-manipulation-striatal-cells-neonatal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.9K 次观看.  National Yang-Ming University. 我们描述了一个立体定向手术的协议, 一个自制的头固定装置 microinjecting 试剂进入新生鼠脑纹状体。这种技术允许在新生小鼠大脑特定区域的神经细胞中进行基因操作。

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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

科研

发育生物学

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

神经科学

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 ...

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

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

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 ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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 ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain regions still presents a challenge, particularly when targeting brain structures along the midline. These challenges include avoiding of the superior sagittal sinus and third ventricle and the ability to consistently target selective and discrete brain nuclei. In addition, more advanced neuroscience techniques (e.g., optogenetics, fiber photometry, and two-photon imaging) rely on targeted implantation of significant hardware to the brain, and spatial limitations are a common hindrance. Presented here is a modifiable protocol for stereotactic targeting of rodent brain structures using an angled coronal approach. It can be adapted to 1) mouse or rat models, 2) various neuroscience techniques, and 3) multiple brain regions. As a representative example, it includes the calculation of stereotactic coordinates for targeting of the mouse hypothalamic ventromedial nucleus (VMN) for an optogenetic inhibition experiment. This procedure begins with the bilateral microinjection of an adeno-associated virus (AAV) encoding a light-sensitive chloride channel (SwiChR++) to a Cre-dependent mouse model, followed by the angled bilateral implantation of fiberoptic cannulae. Using this approach, findings show that activation of a subset of VMN neurons is required for intact glucose counterregulatory responses to insulin-induced hypoglycemia.

Described here is a stereotactic procedure that can target challenging and difficult-to-reach brain regions (due to spatial limitations) using an angled coronal approach. This protocol is adaptable to both mouse and rat models and can be applied to diverse neuroscientific applications, including cannula implantation and microinjections of viral constructs.

多功能神经科学技术的适应性角度立体定向方法

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

<meta charset="utf8"></head><body>化学物质、蛋白质或病毒载体的立体定位颅内递送以研究帕金森病

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

化学物质、蛋白质或病毒载体的立体定位颅内递送以研究帕金森病

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

新生小鼠脑巴细胞基因调控的立体定向手术

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新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用

多功能神经科学技术的适应性角度立体定向方法

化学物质、蛋白质或病毒载体的立体定位颅内递送以研究帕金森病

化学物质、蛋白质或病毒载体的立体定位颅内递送以研究帕金森病