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 border="0" cellpadding="0" cellspacing="0" width="1134">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">30G PrecisionGlide Needle</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">REF 305106</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Chloroform</td>
			<td style="width:273px;">JT Baker</td>
			<td style="width:199px;">9180-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Hamilton MICROLITER Syringe</td>
			<td style="width:273px;">Hamilton&#160;</td>
			<td style="width:199px;">80300</td>
			<td style="width:395px;">30G needle fit for PE10 tube; 26G needle needs a PE20 adaptor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Polyethylene tubing PE20</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">427406</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Polyethylene tubing PE10</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">427401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Micro Flow Rate Syringe Pump</td>
			<td style="width:273px;">Longer Precision Pump Co.</td>
			<td style="width:199px;">TJ-2A (Controller) and L0107-2A (Drive Unit)</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">25G syringe</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">REF 302105</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Fast green</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:199px;">F-7252</td>
			<td style="width:395px;">0.1%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Standard Stereotaxic Instruments</td>
			<td style="width:273px;">RWD Life Science</td>
			<td style="width:199px;">68037</td>
			<td style="width:395px;">Without using 68030 Mouse/Neonatal Rat Adaptor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Anti-FOXP2 antibody</td>
			<td style="width:273px;">Abcam</td>
			<td style="width:199px;">ab16046</td>
			<td style="width:395px;">Rabbit polyclonal to FOXP2, 1:4K</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Anti-RFP antibody</td>
			<td style="width:273px;">Abcam</td>
			<td style="width:199px;">ab65856</td>
			<td style="width:395px;">Mouse monoclonal to RFP, 1:1K</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">BX63 microscope</td>
			<td style="width:273px;">Olympus</td>
			<td style="width:199px;">BX63</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">LSM 880 confocal microscope</td>
			<td style="width:273px;">Zeiss</td>
			<td style="width:199px;">LSM 880</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Goat anti-rabbit conjugated Alexa fluor594</td>
			<td style="width:273px;">Jackson lmmunoReserch Laboratories Inc.</td>
			<td style="width:199px;">111-585-003</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40</td>
			<td style="width:273px;">Penn Vector Core</td>
			<td style="width:199px;">AV-9-PV1848</td>
			<td style="width:395px;">Lot # CS0987, 5.506x1013 (GC/mL)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">AAV9.chicken actin-eGFP</td>
			<td style="width:273px;">AAV core, Institute of Biomedical Sciences, Academia Sinica, Taiwan</td>
			<td style="width:199px;">N/A</td>
			<td style="width:395px;">1x1014 (GC/ml)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:59px;width:269px;">B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td>
			<td style="width:273px;">The Jackson Labtorary&#160;</td>
			<td style="width:199px;">007914</td>
			<td>Ai14</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">B6(Cg)-Foxp2tm1.1Sfis/CfreJ</td>
			<td style="width:273px;">The Jackson Labtorary&#160;</td>
			<td style="width:199px;">026259</td>
			<td style="width:395px;">Foxp2fl/fl</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Dulbecco&#8217;s phosphate buffered saline</td>
			<td style="width:273px;">Corning&#160;cellgro</td>
			<td style="width:199px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

14.8K 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

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

啮齿动物的立体定向手术和动物福利成果改进行为神经科学

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

科研

神经科学

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

小鼠多巴胺和三维胶原基质检测纹状体外植体的解剖和文化

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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 ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

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

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

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

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

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

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

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 ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...