This research was supported by the Robert A. and Rene E. Belfer Family Foundation, NIA R21 AG038856 (JLJ), BrightFocus Foundation Alzheimer&#8217;s Disease research grant A2010097 (JLJ), and NIA Biology of Aging Training grant T32 AG000183 (support for SDG).

执行所有的程序和涉及动物按照健康指南全国学院为护理实验动物的使用和协议。这里所描述的程序进行审查和批准医学实验动物管理和使用委员会的贝勒大学。 在啮齿类动物的脑转基因传送无法复制的腺相关病毒（AAV）载体被批准用于生物安全等级1的使用。请参阅疾病预防控制中心网站，美国政府出版的“生物安全微生物和生物医学实验室（BMBL）”，详细介绍关于妥善保护和病毒处理程序的具体要求。请与当地的兽医和环境安全的工作人员学习​​使用病毒程序制度的具体要求。关于程序的客房，检疫，鉴定和生物危害网箱条例跨机构而有所不同。 1，预削减P0小狗和养母<ol><li>提供高脂食物对所有孕妇和哺乳期的女性，以支持育种和哺乳期的能源需求。 </li><li>到了晚上，成立了养殖笼通过添加一个或两个健康的女性，以一个单一的男性居民的笼子。第二天早上，检查女性的交配塞，并从分离的男性女性，如果一个插件存在。 <ol><li>使用养母从差的产妇特征的遗传背景（例如，C57BL / 6或C3HeJ）提高幼仔。养母接受来自另一窝幼崽，如果转移幼仔在四天之内的养母自己的垃圾诞生。 </li><li>设置使用ICR或FVB雌性一起实验育种，使得两组雌性交付在大约相同的时间独立的交配笼。 </li></ol></li><li>女性将在19±1天内发货从插头日期，根据背景应变。前三天为Delivery（插头日期16天后），把怀孕的女性到一个干净的笼子里有新鲜的筑巢材料和覆盖的栖身之所。 </li><li>检查刚出生的幼崽，每天两次开始前2天预产期（插头日期17天之后）。通过底部的粉色新生儿存在偷看检查笼最小应力的母亲。一般小鼠幼崽传递在早上，但在少数情况下将提供在下午。 </li><li>计划尽快进行注射的幼仔哺乳（这将是可见的牛奶斑明显），或在6小时出生，以先到者为准。 </li></ol> 2，备好设备注射<ol><li>准备一个10微升注射器用32克的针一般P0新生儿。如果进行立体定位注射，装入注射器上的机械臂。 </li><li>如果使用的是立体框架注射，冷却新生儿阶段4-8℃，加入100％伊桑醇和干冰到储存在该块的前端。保持高于1℃的温度，以避免幼仔的冻伤。 </li></ol> 3，准备稀释病毒注射<ol><li>准备一个1％台盼蓝染料溶液（20倍的股票）。 </li><li>准备的腺相关病毒（AAV）股票5-25微升等份2级生物安全柜内。将这些等分试样在-80℃下以供将来使用。避免额外的冻融循环，因为这会导致病毒失去传导的功效。 </li><li>从-80℃冰箱中，置于冰上取下的AAV等分解冻。 </li><li>通过稀释的病毒入冰冷的1X磷酸盐缓冲盐水（PBS）中制备的病毒溶液用于注射。开始用10倍连续稀释（十月10日至8月 10日的病毒颗粒/半球）的转导模式的初始优化。 </li><li>加入20倍的台盼蓝对AAV溶液至0.05％的最终浓度。 </li></ol><pCLASS =“jove_title”&gt; 4。举办​​新生幼犬注射<ol><li>置于冰上小铝板冷却。将干燥的任务擦上盘的顶部，以保护小狗的皮肤从冰冷的金属。这就像一台冰冷的表面进行麻醉和注射的幼崽。 </li><li>准备适合饲养新生小鼠温暖注射前后变暖垫。 </li><li>一旦新生幼崽已开始吃奶，从笼子里取出大约一半的人留下的另一半与他们的母亲。将收集到的幼崽在变暖垫在等待注射。 注意：如果亲生母亲不关心幼仔，从笼子里立即删除整个垃圾，并把幼崽到暖垫等候注射。这种情况可能出现的出生对C57BL / 6的女性，或与具有较差特色养殖等近交系的第一胎。在这种情况下，牛奶斑不会对新生幼仔可见。 </li><li>传输距离变暖垫1的小狗到冰冷的金属板，诱导低温麻醉。等待2-3分钟的小狗变得完全麻醉。通过轻轻地挤压爪子确认麻醉和监测缺乏运动或呼吸。 </li></ol> 5，注射AAV到新生小鼠<ol><li>徒手颅内注射AAV到新生小鼠： <ol><li>负载5微升稀释的AAV用0.05％台盼蓝入注射器。 </li><li>轻轻擦拭麻醉的小狗的头，用棉签浸在70％的乙醇。 </li><li>确定注射部位处从拉姆达缝合到每只眼睛的距离五分之二。另一种注射部位位于约0.8-1毫米的矢状缝外侧，中途Lambda和前囟门之间。这些标志性建筑都可见通过皮肤在P0。 </li><li>马克注射部位与无毒的实验室笔。 </li><li>保持与规模MONI可见注射器toring的溶液的体积分配。 </li><li>确保拇指能达到柱塞的顶部。然后从柱塞虽然定位针，以避免意外配药病毒移除大拇指。 </li><li>找到一个舒适的位置，通过将板凳上的肘部和靠在手臂上的冰桶，以振奋的手臂注射。 </li><li>侧面朝下放置，其头部的小狗直属注射器。把小狗的头，使标记的注射部位朝上，轻轻地但坚定地保持这个姿势一条生路。 </li><li>持注射器垂直于颅骨的表面和插入针在标记的注射部位到约3mm的深度。注射针，直到阻力略有降低，表明针头已渗透到侧脑室。如果需要的参考，可3毫米针具有无毒制造的尖端。调整基于染料吨注射深度为小狗的大小和应变argeting心室。 </li><li>持注射器刚性，使柱塞可以是凹陷而不更远移动针进入大脑。如果注射移位到丘脑，病毒传播将受到严重的限制。 </li><li>开始慢慢注入病毒，同时监测从注射器分配的体积。辖2微升，最大容量为每一个心室。如果针处于正确的位置时，染料会扩散到填充心室。 </li><li>慢慢地拔出针头。 </li><li>允许第一注射部位以关闭注入另一半球之前。 注意：不要注射过一次同一站点多。重新插入针力病毒已经注入漏出，造成伤害的小狗或死亡。 </li><li>注射用同样的步骤对侧心室。 </li></ol></li><li>立体定向注射AAV到新生小鼠： <ol><li>负载5微升稀释的AAV用0.05％台盼蓝整数Ø通过立体操盘举行的注射器。 </li><li>轻轻将小狗的头新生儿帧的耳棒之间。确保磁头是通过检查拉姆达和囟门之间的线平行于台在Y轴的水平（从前到后）。确保磁头是通过检查耳朵或眼睛之间的线之间的想像线，平行于该阶段中的X轴水平（从一侧到另一侧）。 </li><li>轻轻擦拭麻醉的小狗的头，用棉签浸泡于70％乙醇。 </li><li>使用立体定向操纵器来定位上面的λ的注射器，然后调零的X和Y坐标。将立体手臂（X，Y）=（0.8，1.5）毫米标准P0幼仔。 注：小狗的尺寸和立体坐标可以通过应变和年龄而有所不同。根据需要对目标侧脑室调整基于染料注射坐标。 </li><li>慢慢放下针头插入注射部位。头骨的表面会IND耳鼻喉科，然后释放，一旦针头穿透皮肤。收回该针头至颅骨恢复其正常凹的形状，但保持所述针的斜面的皮肤下。零，在这点的Z坐标。 </li><li>将针头至Z = -1.7毫米，然后缩回至-1.5毫米。 </li><li>慢慢地注入病毒。每个半球最多可以接受2微升的解决方案。 </li><li>保持就位的注射器30-60秒完成注射后，然后缓慢缩回针超过1-2分钟。 </li><li>重复对侧半球，在X轴为注射部位使用负坐标。 请注意：用于注射的替代的坐标（X，Y，Z）=（0.8，2.0，-​​1.5）mm和（1.2，1.0，-1.5）毫米。执行第一次注射（0.8，2.0），然后移动到（0.8，-2.0），（1.2，1.0），最后（1.2，-1.0）。注入1微升的溶液在每个站点，总共每半球2微升。快速移动的注射完成在Les程序总比10分钟。 </li></ol></li></ol> 6，注射后护理<ol><li>完成注射到两个半球后，将小狗放回变暖垫，直到其体温和皮肤颜色恢复正常和小狗开始移动。 </li><li>如果使用的是亲生母亲喂养的新生儿注射，恢复注入幼崽的生母他们恢复正常运动后。将剩余的未注射的幼崽在变暖垫。重复上述步骤，直到所有的老鼠都被注射。 </li><li>如果使用的是寄养妈妈喂养的新生儿注射，注射的所有幼崽转移到养母照顾他们恢复正常运动后。 <ol><li>除去大部分或全部从笼属于养母幼仔的，以确保注射的动物的成功。 </li><li>如果寄养母亲的幼崽，并注射幼崽有相同的眼睛和皮肤的颜色，标记从养母幼仔用实验室的笔，使他们能够后来区分。 </li><li>将养母的幼崽和他们的一些床上用品一起注入幼崽。确保注入的幼崽获得她的亲生子女的香味，提高接受新的幼崽。 </li><li>将只注入幼崽放回养母的笼子。剔除从养母任何剩余的幼崽。 </li></ol></li><li>确保母亲抓和哺乳把它们入笼的注入幼仔在10分钟内。如果母亲没有在这段时间内收集的幼崽，幼崽转移到另一个的养母。 </li><li>标签笼用生物危害卡以显示它含有病毒注入动物。众议院在72小时经批准的区域内笼。 </li><li>在72小时的检疫期后，母亲和幼仔（但没有床上用品或其他物品笼）转移到一个干净的笼子。执行此转让2级生物安全柜内，以免接触到任何病毒污染的床上用品。 ŘE打开笼子的清洁与动物注射到普通的鼠标设备。 </li><li>将脏笼成生物危害袋，通过高压灭菌消毒，然后将保持架返回到动物饲养。 </li></ol> 7清理<ol><li>将剩余的病毒溶液，在4℃以备将来使用。病毒保留的转导效率为至少2个月贮存于4℃下8时。 </li><li>消毒的注射针和注射器，用2％漂白剂随后在去离子水中反复冲洗。 </li><li>清洗，用2％漂白接着用70％乙醇在工作区中。 </li><li>收集所有的一次性项目，包括吸头，管，口罩和手套在一个塑料生物危害袋。按照当地的法律和制度的政策（ 即高压灭菌或焚烧）处置废料。 </li></ol> 8，准备老鼠大脑成像<ol><li>将注入小鼠成二氧化碳安乐死室3处后4周注射。遵循正确的安乐死程序制度指引。不要灌注动物与多聚甲醛，因为这会熄灭的荧光标记。 </li><li>取下头盖骨的整个大脑，并在4℃定为3-12小时，在PBS中的4％多聚甲醛。 </li><li>固定脑转移到30％蔗糖的冷冻保护。孵育在4℃下直到大脑下沉到水底。 </li><li>收集细碎的干冰一桶。切开大脑半年下来中线。埋半脑在干冰冻结。 </li><li>通过加入干冰和100％的乙醇冷却切片机的阶段。 </li><li>保护大脑与中线向下使用PBS的阶段，并允许冻结。 </li><li>用冷冻滑动切片机通过大脑科30-45微米厚。 </li><li>处的部分为含有防冻剂溶液（250毫升甘油300毫升乙二醇500毫升的0.1M磷酸盐缓冲液，pH 7.4）的24孔板。盖板无线第锡箔，并储存在-20℃下以供将来使用。 </li><li> 1-4井收集部分，并在PBS冲洗3次。 </li><li>安装部分到显微镜载玻片和盖玻片。让幻灯片干在黑暗的位置。 </li><li>使用适当的过滤器原生YFP或tdTomato图片幻灯片，检查病毒转导的模式。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

我们已经描述了一种通用的程序，用于操纵使用AAV作为广泛递送到新生小鼠脑部的车辆神经元基因表达。与神经元的转基因的其它方法，如在子宫内电1或立体定向颅内注射2,3相比，新生病毒注射是比较容易和简单。基本过程可以在几分钟内完成，只有一个冰桶和微升注射器。最佳的生存和转基因表达可以通过参加几个技术细节来实现，最重要的是病毒性的股票，时机和注射的准?...

传统的方法用于修改神经基因表达需要耗时和昂贵的种系操作。另一种从头方法，如在子宫内电穿孔或立体定位慢病毒注射液产生更快的结果，而且成本较低，但有需要复杂的外科手术1-3的劣势。此外，转基因表达具有有限的空间范围使用这些方法。在此，我们通过脑室注射腺相关病毒（AAV）到新生小鼠大脑描述了一种快速，简便，经济的方法进行广泛的神经元处理。该方法最初是由约翰·沃尔夫和Marco Passini于2001年，在那里他们建议的小粒径的AAV允许它在脑脊髓液中的扩散，因为它从侧脑室穿过未成熟的室管膜屏障并进入脑实质4描述的， 5。脑室注射腺相关病毒的F内IRST的出生国债收益率神经亚群跨越大脑的各个区域，从嗅球脑干6,7普遍病毒转导后24小时。病毒交付的转基因表达，并在注射几天活跃，持续长达一年后转。因此，这种多功能的操作使研究从出生后早期大脑发育到衰老和退变的成人。 在适应的技术，我们的具体实验需要，我们主要侧重于AAV8血清型，因为它是最有效的传感神经元6。我们表明，病毒滴度可以稀释，以控制转导神经细胞的遗传操作的实验检测细胞内在后果的密度。此外，我们证明，这两种病毒可以被共注射，以产生被偏向不同的或重叠的集合的神经元的表达模式，这取决于所选择的血清型病毒包装。我们的工作扩展这种技术在范围广泛的实验神经科学设置使用的通用性。

<table border="0" cellpadding="0" cellspacing="0" width="564">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Name of Material/ Equipment</td>
			<td style="width:89px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">ICR outbred mice</td>
			<td style="width:89px;">Harlan</td>
			<td style="width:119px;">Hsd:ICR (CD-1)</td>
			<td style="width:167px;">This strain is also known as CD-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">FVB inbred mice</td>
			<td style="width:89px;">The Jackson Laboratory</td>
			<td style="width:119px;">1800</td>
			<td style="width:167px;">5-6 weeks of age</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Nestlets</td>
			<td style="width:89px;">Lab Supply</td>
			<td style="width:119px;">NESTLETS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Shepherd shacks</td>
			<td style="width:89px;">Lab Supply</td>
			<td style="width:119px;">SS-mouse</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">High fat rodent chow</td>
			<td style="width:89px;">Purina Mills</td>
			<td style="width:119px;">PicoLab Mouse diet 20, #5058</td>
			<td style="width:167px;">This is our standard breeder chow</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:189px;">High fat rodent chow (alternative)</td>
			<td style="width:89px;">Harlan Laboratories</td>
			<td style="width:119px;">Teklad Global 19% protein rodent diet #2019S</td>
			<td style="width:167px;">If low phytoestrogen, autoclavable diet is needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Injection syringe</td>
			<td style="width:89px;">Hamilton</td>
			<td style="width:119px;">7653-01</td>
			<td style="width:167px;">10 ml syringe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Injection needles</td>
			<td style="width:89px;">Hamilton</td>
			<td style="width:119px;">7803-04, RN 6PK PT4</td>
			<td style="width:167px;">32 gauge, for standard P0 injections</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:189px;">Metal plate for cryoanesthesia</td>
			<td style="width:89px;">McMaster Carr</td>
			<td style="width:119px;">8975K439</td>
			<td style="width:167px;">Raw aluminum plate, 6&#8221; x 12&#8221;, 0.25&#8221; thick, will need to be cut into 3 equal pieces and edges sanded by local machine shop</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Small animal stereotaxic device with digital readout</td>
			<td style="width:89px;">David Kopf Instruments</td>
			<td style="width:119px;">Model 940</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Universal syringe holder with needle support foot</td>
			<td style="width:89px;">David Kopf Instruments</td>
			<td style="width:119px;">Model 1772-F1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Neonatal frame</td>
			<td style="width:89px;">Stoelting</td>
			<td style="width:119px;">51625</td>
			<td style="width:167px;">Officially called a mouse and neonatal rat adaptor</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:189px;">Biohazard disposal bags with sterile indicator</td>
			<td style="width:89px;">VWR</td>
			<td style="width:119px;">14220-030</td>
			<td style="width:167px;">Important! &#8211; Check with local veterinary and environmental safety staff to learn your institute&#8217;s protocol for biohazard waste disposal</td>
		</tr>
	</tbody>
</table>

intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction

随着科技进步的步伐迅速加快，需要对实验神经科学的新方法，能够快速，轻松地操纵基因表达的小鼠大脑。在这里，我们描述了一种技术，最早由Passini和沃尔夫介绍了颅内直接供货病毒编码的转基因到新生小鼠大脑。在其最基本的形式中，该过程仅需要一个冰桶和微升注射器。然而，该协议也可适于与立体定位框架上使用，以提高用于研究新的技术一致。该方法依赖于重组腺相关病毒（AAV），以从脑室自由移动进入脑实质，而室管膜衬里仍然是在出生后的第12-24小时期间，未成熟的能力。脑室注射的AAV在这个年龄导致的神经元在整个大脑的普遍转导。在注射几天表达开始，持续了lifetimE中的动物。病毒滴度可以调节，以控制转导神经元的密度，而共表达的荧光蛋白的规定转导的细胞的一个重要的标签。随着病毒核心设施的上升可用性，以提供现成的货架，预包装试剂和自定义病毒制剂，这种方法提供了及时的方法在小鼠大脑中快速，方便，便宜得多操纵基因表达比传统的生殖工程。

成功脑室病毒注射液产量普遍和强大的神经元表达。在这里，我们评估病毒转导使用YFP或鸡β肌动蛋白启动子（CBA子）的控制下tdTomato荧光基因。这些构建体包装到AAV8和注入新生（P0），ICR小鼠的侧脑室。高病毒效价（每半球10 10个粒子）导致致密标签嗅球，纹状体，皮层，海马和小脑（ 图1A，左）。标签也是在小脑浦肯野神经元明显，但小脑颗粒神经元的尚未出现大量的P0明?...

Watch this Scientific Journal Video about Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction at JoVE.com

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

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 ...

intracerebroventricular-viral-injection-neonatal-mouse-brain-for

Research

JoVE Journal

Neuroscience

57.8K 次观看.  Baylor College of Medicine. 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.

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

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

科研

神经科学

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 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 and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

小鼠新生儿缺氧缺血性脑损伤模型的神经行为评估

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

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, ...

Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric spinal cord injuries (SCIs) compared to adult SCIs. In addition, reliably introducing therapies into the target cells of the central nervous system (CNS) can be challenging, and inaccuracies can compromise the efficacy of the study or therapy. This protocol combines viral vector technology with a novel surgical technique&#160;to accurately introduce gene therapies into neonatal rats at postnatal day 5. Here, a virus engineered for retrograde transport (retroAAV2) of Cre is introduced at the axon terminals of corticospinal neurons in the spinal cord, where it is subsequently transported to the cell bodies. A double-floxed&#160;inverted orientation (DIO) designer receptor exclusively activated by designer drug(s) (DREADD) virus is then injected into the somatomotor cortex of the brain. This double-infection technique promotes the expression of the DREADDs only in the co-infected corticospinal tract (CST) neurons.&#160;Thus, the simultaneous co-injection of the somatomotor cortex and cervical CST terminals is a valid method for studying the chemogenetic modulation of recovery following cervical SCI models in neonatal rats.

This protocol demonstrates a novel method for applying gene therapies to subpopulations of cells in neonatal rats at postnatal ages 5-10 days by injecting an anterograde chemogenetic modifier into the somatomotor cortex and a retrogradely transportable Cre recombinase into the cervical spinal cord.

使用脑和脊柱联合手术用双病毒载体靶向新生大鼠的皮质脊髓束

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

立体定向病毒注射和梯度指数晶状体植入术用于深部脑 体内 钙成像

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

强大且高度可重复的皮质脑类器官生成，用于在体外模拟大脑神经元衰老

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...