国家自然科学基金（授予孙晓彦82101249项），国家博士后科研工作基金（授予孙晓彦，批准号:2022M722125授予孙晓彦）。上海市帆船项目（授权号:21YF1425100）上海市卫生健康委员会临床研究专项（J 周 202340088 号拨款）.国家自然科学基金（第82101262项授予张旭，第82101287项授予陈旭）。

所有动物实验均按照《动物研究报告体内实验》（ARRIVE）指南和美国国立卫生研究院《实验动物护理和使用指南》进行。本研究经上海交通大学医学院附属仁济医院动物护理与使用专业委员会批准。本研究采用8周龄C57BL/6J雄性小鼠。这些动物是商业获得的（见 材料表）并饲养在标准笼子中（22°C±2°C，12小时/ 12小时光照/黑暗循环，食物和水随意）。
1. 目标脑区域的选择
注意:这是一个无菌程序。确保所有手术器械都经过消毒。在接触手术器械之前，用酒精喷洒手套。避免戴手套触摸器械尖端或手术区域。在开始手术之前，通过检查矫正反射的丧失和对皮肤刺激没有反应，确保小鼠得到充分麻醉。在钻孔和注射之前，密切监测呼吸频率（每分钟60-220次呼吸，平均每分钟100-150次呼吸）和呼吸幅度（应保持一致，不要过浅或过深）。应注意呼吸频率或振幅的任何显着变化。
<ol><li>使用29G针头腹膜内注射1.25%三溴乙醇（250mg / kg）麻醉小鼠。一旦矫正反射消失，就剃掉头骨上的毛发。用鼻夹固定上门牙并使用两个耳杆稳定头部，将鼠标固定在立体定位框架中（参见 材料表）。</li>
	<li>使用眼药膏以防止眼睛干燥。用 Anerdian（消毒溶液;见 材料表）擦拭头皮三次对头皮进行消毒。使用 29 G 胰岛素针头皮下注射 0.2 mL 1% 利多卡因以提供局部镇痛。</li>
	<li>用眼剪刀做一个 0.5 厘米的切口，露出颅骨。使用蘸有 3% H2O2 的棉签擦洗暴露的颅骨以去除骨膜。让颅骨干燥。</li>
	<li>将钻头固定在立体定位仪器上。借助放大镜调整立体定位臂的旋钮，将钻尖精确定位在前囟上，并使用 LCD 数字显示屏记录 z 轴值。<ol><li>调整旋钮并将钻头放置在 lambda 上并记录 z 轴的值。调整鼻夹和耳杆，使两个 z 值更近，然后重复上述步骤，直到两个 z 值之间的差异小于 0.1 mm。 注意: LCD 数字显示屏上的 x、y 和 z 轴分别对应于内外侧 （ML）、前后 （AP） 和背腹 （DV）。一旦两个 z 值之间的差值小于 0.1 mm，就可以认为 bregma 和 lambda 位于同一水平面上。</li>
	</ol></li>
	<li>确保大脑的左右水平度。使用相同的刻度调整连接到立体定位框架的耳杆。以侧齿被盖腹侧部分（LDTgV）为例;根据 Paxinos 和富兰克林的小鼠大脑图谱16，它的立体定位坐标为 -5.2 表示 x，+0.8 表示 y，表示 z。<ol><li>调整耳杆，使钻头在（-5.2，+0.8）处测量背腹方向的z值，将钻头移动到（-5.2，-0.8）处测量z值。 注意: 当两个 z 值之间的差值小于 0.2 毫米时，左右被视为处于相等水平。耳杆需要插入正确的位置，因为外耳道是对称的。如果两个 z 值之间的差异超过 0.5 毫米，则耳杆可能插入了错误的位置。</li>
	</ol></li>
	<li>再次将钻头定位到前门，并在 LCD 数字显示屏上将三个坐标设置为零。使用参考坐标将钻头放置在感兴趣区域上方。开始演练。<ol><li>使用垂直臂逐渐降低钻头，直到它穿透颅骨。用棉签清除碎屑和血液。 注意:注意不要损伤脑组织。</li>
	</ol></li>
</ol>2.染料溶液的制备和脑显微注射
<ol><li>将 25 μL 含有溴酚蓝的 SDS-PAGE 上样缓冲液（参见 材料表）放入 200 μL 微量离心管中。向试管中加入 50 μL ddH2O 以制备染料溶液。</li>
	<li>用盐水湿的化妆棉盖住颅窗。通过反复抽吸和排出生理盐水来确保微注射器通畅。将 0.3 μL 染料加载到微量注射器中。将注射器和电动立体定位显微注射器连接到立体定位臂上。</li>
	<li>将注射器尖端放在前囟处，并在 LCD 数字显示屏上将坐标设置为零。根据参考坐标将针头调整到感兴趣的区域。将进样速度设置为 0.1 μL/min，然后启动电动显微注射器，将 0.3 μL 蓝色染料输送到目标区域。将注射器保持在原位至少 10 分钟，然后慢慢抽出注射器。 注意:在同一只小鼠中可以使用不同的颜色染料来区分紧密定位的区域。根据其他染料的物理特性测试其合适的浓度。建议使用溴酚蓝配方，并在使用前后彻底冲洗注射器，以防止堵塞。根据染料扩散调整进样体积。</li>
</ol>3. 脑组织采集和冷冻切片
<ol><li>将鼠标从立体定位帧中释放出来。使用29G针头腹膜内注射50mg / kg戊巴比妥钠麻醉小鼠。用胶带将鼠标固定在泡沫板上。</li>
	<li>打开胸部两侧以引起快速窒息。在右心耳上做一个 1-2 毫米的切口。将输液针插入左心室。输注 20 mL 预冷盐水和 20 mL 冰镇 4% 多聚甲醛溶液。</li>
	<li>用组织解剖剪刀斩首小鼠（遵循机构批准的方案）。从颈部到鼻子的中线切开一个切口，去除皮肤，然后露出头骨。用镊子或剪刀清除颅骨上的残留肌肉。</li>
	<li>将细剪刀的一根刀片的尖端放在枕骨大孔和大脑之间，锋利的边缘朝向骨头，位于矢状缝合线中部的喙端。继续沿着中矢状缝合线滑动并切割颅骨。用镊子切除头骨，露出暴露的大脑。 注意: 请注意，以免在手术过程中对大脑造成任何伤害。在取出骨屑之前，小心地剥去可能附着在颅骨上的任何脑膜。这种预防措施对于防止在此过程中无意中切开脑组织至关重要。</li>
	<li>打开旋转式切片机低温恒温器的电源（参见 材料表）并将腔室温度设置为 -20 °C。 倒入少许 OCT 化合物，均匀地覆盖标本盘的表面。将其放入冷冻室中，让 OCT 凝固。</li>
	<li>将圆盘连接到标本头上。将刀片靠近块，并修剪 OCT 块以形成平行于圆盘的平面。</li>
	<li>用剃须刀片在脑切片模具中，将大脑沿冠状方向切开，远离注射部位。将包含注射部位的脑组织，切口面朝下，放在 OCT 平面上。<ol><li>将 OCT 倒入大脑中，然后将圆盘放入冷冻室，让标本块凝固。将 OCT 反复倒入大脑，直到大脑完全嵌入 OCT。 注意:逐层添加 OCT 化合物。一旦前一层 OCT 被冻结，将下一层 OCT 化合物倒入大脑。</li>
	</ol></li>
	<li>修剪试样块，直到目标区域接近。从注射部位级别开始制作几个部分。使用保持在-20°C的小书写刷将冠状脑切片收集到含有室温PBS的6孔板中。 注意: 将切割厚度设置为更大的厚度以修剪块，然后在刀片接近注射部位时将切割厚度设置为 40 μm。如果使用神经回路示踪剂，则不会立即处死小鼠。将小鼠置于加热毯上的无菌术后恢复笼中，以便从麻醉中恢复。一旦醒来，将小鼠放回原来的笼子或新的笼子。</li>
</ol>4. 影像学检查
<ol><li>用室温PBS洗涤OCT。用刷子捡起大脑部分并将它们放在载玻片上。让切片在室温下风干。</li>
	<li>目视观察注射区域，当目标细胞核很小时，使用显微镜观察。将注射染料的位置与大脑图谱中的相应区域进行比较。 注意: 根据注射点偏差的方向和距离调整 x、y 和 z 轴的值。可能需要多次重复此协议中的所有步骤才能准确确定坐标。</li>
</ol>

优化小鼠立体定向显微注射的准确性

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提交人声明没有竞争利益。

本文描述了一种稳定的策略，可以在病毒追踪之前更快、更简单地验证立体定向脑注射的准确性 5,6，但报告基因在大脑区域表达的不可替代性对于大脑区域标记至关重要。我们使用的蓝色染料可以立即看到注射部位。
该协议中的几个关键步骤有助于提高注射部位的准确性。首先，从步骤1.5开始，确保大脑在立体定位仪中的左右水?...

几乎所有现代神经调控工具，包括体内钙记录、光遗传学和化学遗传学工具，都需要使用立体定位坐标来靶向感兴趣的大脑区域 1,2,3，从而形成神经操作的基础。小鼠大脑区域的立体定位坐标是相对于前囟和λ（颅骨上的骨标志）定义的，形成了所谓的颅骨衍生的立体定位坐标系统。bregma 或 lambda 都可以用作三维坐标的零点。这三个轴是前后轴 （AP）、内外侧轴 （ML） 和背腹轴 （DV），代表立体定位仪器数字显示屏上的 y、x 和 z 轴。对于众所周知的大脑区域，特定区域的立体定位坐标参数可以从小鼠脑图谱4（例如，Paxinos和Franklin的小鼠大脑在立体定位坐标中）和/或已发表的文献5,6中获得。然而，由于立体定位设备的变化和不同研究人员使用的小鼠的年龄/群体，需要进一步验证。
结构是功能的基础。神经回路构成了许多大脑功能的基础，例如认知活动、情绪、记忆、感觉和运动功能1.标记结构和操纵神经回路的活动对于理解特定神经回路的功能至关重要。在过去的几十年里，神经示踪剂已经进化了好几代;早期研究采用小麦胚芽凝集素（WGA）和菜豆凝集素（PHA）作为顺行示踪剂，氟金（FG）、霍乱毒素B亚基（CTB）、痰花青作为逆行示踪剂。然而，与病毒示踪剂不同，这些传统的神经示踪剂不能将外源基因整合到宿主中，也不具有细胞类型选择性。如今，病毒策略已成为基础神经科学研究中的一个重要命题。针对不同的研究目的，可以选择各种病毒工具7,8。有非跨突触病毒、反式多突触病毒（逆行和顺行）和跨单突触病毒（逆行和顺行）。每个类别都包含几种具有各自特征的类型。
病毒给药和表达的过程非常耗费时间和资源，通常需要数周甚至更长时间。在各种病毒载体中，腺相关病毒已被确定为一种有前途的基因递送手段，为实验程序提供了注射后 3 至 8 周的广泛窗口 7,8。随着 AAV 的发展，可以在给药后 2-3 周进行分析 9,10。其他神经回路示踪剂，如伪狂犬病病毒 （PRV） 和狂犬病病毒 （RV），也需要至少 2-7 天的追踪期 11,12,13,14,15。因此，在观察荧光信号之前对注射部位进行初步验证既省时又经济。
为了便于简单快速地验证立体定向注射，在这项研究中，在病毒载体之前施用染料，冷冻切片使研究人员能够在注射后 30 分钟内观察注射部位并对其进行追踪。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.0 &#181;L, Neuros Syringe, Model 7001 KH, 32 G, Point Style 3</td>
			<td>Hamilton</td>
			<td>65458-01</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L pipette tips</td>
			<td>biosharp</td>
			<td>BS-200-T</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL syringe</td>
			<td>Kindly group</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3%H2O2 solution</td>
			<td>Lircon Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Shengyou Biotech</td>
			<td>20006</td>
			<td></td>
		</tr>
		<tr>
			<td>Anerdian</td>
			<td>Likang High-tech</td>
			<td>31001002</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti roll plate</td>
			<td>Leica</td>
			<td>14047742497</td>
			<td></td>
		</tr>
		<tr>
			<td>BD insulin syringe</td>
			<td>Becton,Dickinson and Company</td>
			<td>328421</td>
			<td></td>
		</tr>
		<tr>
			<td>Bend toothed dissecting forceps</td>
			<td>Jinzhong</td>
			<td>JD1050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellsens dimension software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Fisher Scientific</td>
			<td>23-400-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi-Fluoromount-G</td>
			<td>Southernbiotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Longxiang</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine brushes</td>
			<td>HWAHONG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>Jinzhong</td>
			<td>y00030</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscopy</td>
			<td>Olympus</td>
			<td>BX63</td>
			<td></td>
		</tr>
		<tr>
			<td>freezing microtome</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic forceps straight with tooth</td>
			<td>Jinzhong</td>
			<td>J31010</td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion needle 0.7 mm</td>
			<td>Kindly group</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine hydrochloride injection</td>
			<td>Harvest Pharmaceutical Company</td>
			<td>71230803</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnifying glass</td>
			<td>M&#38;G Chenguang Stationery</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Male C57/BL mice</td>
			<td>The Shanghai Institute of Planned Parenthood Research&#8211;BK Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mice coronal brain slice mold</td>
			<td>RWD Life Science</td>
			<td>68713</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>biosharp</td>
			<td>BS-02-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blades</td>
			<td>Leica</td>
			<td>819</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Cisen Pharmaceutical Company</td>
			<td>G23HDM9M4S5</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Biosharp</td>
			<td>BL539A</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pumps</td>
			<td>Harvard Apparatus</td>
			<td>70-4507</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Servicebio</td>
			<td>G4202</td>
			<td></td>
		</tr>
		<tr>
			<td>Piette 2-200 &#956;L</td>
			<td>thermofisher</td>
			<td>4642080</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE sample loading containing bromophenol blue</td>
			<td>Beyotime</td>
			<td>P0015A</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaving blades</td>
			<td>BFYING</td>
			<td>91560618</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Citotest Scientific</td>
			<td>188105</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>RWD Life Science</td>
			<td>68807</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight toothed dissecting forceps</td>
			<td>Jinzhong</td>
			<td>JD1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Holder</td>
			<td>RWD Life Science</td>
			<td>68206</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>Jinzhong</td>
			<td>J21040</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>Aibei Biotechnology</td>
			<td>M2910</td>
			<td></td>
		</tr>
	</tbody>
</table>

preliminary validation of stereotaxic injection coordinates via cryosectioning

特定大脑区域的立体定向注射构成了基础神经科学中的一项基本实验技术。研究人员通常根据小鼠脑图谱或已发表的材料来选择立体定位注射参数，这些材料采用了不同群体/年龄的小鼠和不同的立体定位设备，因此需要进一步验证立体定位坐标参数。钙成像、化学遗传学和光遗传学操作的功效依赖于感兴趣区域内报告基因的精确表达，通常需要数周的努力。因此，如果不事先验证目标大脑区域的坐标，这将是一项耗时的任务。使用适当的染料代替病毒并实施冷冻切片，研究人员可以在染料给药后立即观察注射部位。这有助于在实际注射位置和理论位置之间存在差异的情况下及时调整坐标参数。在随后的实验中，这种调整显著提高了靶区域内病毒表达的准确性。

该研究使用演示的方法在 30 分钟内成功确定了注射部位。最初，将含有溴酚蓝的SDS-PAGE样品上样溶液注射到C57 / BL小鼠的LDTgV中。 图1A 显示了染料溶液注入的示意图。蓝色染料在LDTgV中的分布如图 1B所示。
还将溴酚蓝注射到 mPFC 前边缘皮层、mPFC 边缘下皮层和基底杏仁核 （BMA） 中，以评估该方案的普遍性。如图1C-E</...

作者聚焦:通过立体定向注射和经济高效的染料加速小鼠认知障碍研究

本方案描述了一种实用策略，用于在使用染料和冷冻切片进行病毒示踪之前加快立体定位注射坐标的验证步骤。

通过冷冻切片初步验证立体定向注射坐标

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their ...

我们的研究主要集中在研究小鼠认知障碍的神经回路机制。在这些研究中，我们经常采用大脑立体定位技术。我们的目标是优化和加速使用染料的立体定向注射位点的验证过程，确保它成为一个简单快速的过程。通过立体定向注射病毒，各种策略在神经科学研究中变得至关重要。接下来是观察组织切片中的荧光标记物以确认注射部位，这一过程需要数周时间才能完成。与其他技术相比，我们的方案具有优势，可节省至少三天的脱水时间，并使用具有成本效益的染料更快地识别注射部位。与没有初步验证相比，它可以节省数周时间。

preliminary-validation-stereotaxic-injection-coordinates-via

Research

JoVE Journal

Neuroscience

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

1.3K 次观看.  Shanghai Jiaotong University, School of Medicine. 我们的研究主要集中在研究小鼠认知障碍的神经回路机制。在这些研究中，我们经常采用大脑立体定位技术。我们的目标是优化和加速使用染料的立体定向注射位点的验证过程，确保它成为一个简单快速的过程。通过立体定向注射病毒，各种策略在神经科学研究中变得至关重要。接下来是观察组织切片中的荧光标记物以确认注射部位，这一过程需要数周时间才能完成。?...

视频: 作者聚焦:通过立体定向注射和经济高效的染料加速小鼠认知障碍研究

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 ...

科研

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 ...

Building An Open-source Robotic Stereotaxic Instrument

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The robot&#39;s flexibility and open coding system (g-code) make it capable of performing custom tasks that are not supported by commercial systems. Its applications include, but are not limited to, drilling holes, sharp edge craniotomies, skull thinning, and lowering electrodes or cannula. In order to expedite the writing of g-coding for simple surgeries, we have developed custom scripts that allow individuals to design a surgery with no knowledge of programming. However, for users to get the most out of the motorized stereotax, it would be beneficial to be knowledgeable in mathematical programming and G-Coding (simple programming for CNC machining).
The recommended drill speed is greater than 40,000 rpm. The stepper motor resolution is 1.8&#176;/Step, geared to 0.346&#176;/Step. A standard stereotax has a resolution of 2.88 &#956;m/step. The maximum recommended cutting speed is 500 &#956;m/sec. The maximum recommended jogging speed is 3,500 &#956;m/sec. The maximum recommended drill bit size is HP 2.

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

建设一个开放源码的机器人立体定位仪

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

用于访问视网膜下腔的另类和验证注射法<i&gt;通过</i&gt;一经巩膜后路

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

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, ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

多鼠脑的同时 Cryosectioning

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).