这项工作是由来自美国国立卫生研究院研究所（NINDS）RO1的赠款支持。

1。准备用于注射的病毒<ol><li> rAAVs均购自商业供应商的建议，但他们也可以在自己的实验室中产生的（见下面的讨论）。 〜1 × 12，每毫升的基因组副本（GC /毫升）的滴度和病毒解决方案通常是生产，可在全滴度用来处理大量的细胞。此外，他们可能会被摊薄产生稀疏标签的理想水平。适当稀释，必须由用户决定，但建议开始1:10稀释。 </li><li>使用前在冰上解冻病毒。转移到一个小（〜250μl）PCR管中的病毒解决方案所需的预稀释量。稀释病毒的解决方案，使用适当的音量在组织培养罩无菌DPBS。存储在冰块稀释病毒解决方案，直到需要它。 </li></ol>注意:处理所有病毒，应按照您的机构批准的与生物安全议定书进行 2。注射器装载和设备组装<ol><li>加载针连接的玻璃注射器和PCR管的病毒解决方案，轻轻拉出柱塞。拉出柱塞，直到注射器可以看到一个非常小的泡沫，空气，这表明，所有的解决方案是在注射器中，并没有留在装载针。此外，少量的惰性染料可以被添加到解决方案，以使其可见。 注:应加载针通常没有足够长的时间，以达到大多数其它管子的底部使用一个小的PCR管。 </li><li>卸下装载的的针头和地方注射器注射泵块注射器，安全注射器固定。 </li><li>轻轻插入到注射器的连接管的一端插入另一端的持针器。插入注射针持针器。注射针到底应该直接接触的持针器的连接管内结束。 </li><li>慢慢地压低柱塞手动推通过连接管的解决方案。推，直到小滴的解决方案是在注射针的尖端可见。 </li><li>仔细柱塞推块的位置和推块支架的地方，它的安全。 </li><li>设定注射参数:注射速度应约8μl/min（〜130nl / S）;注射量一般为1 - 2ul;选择适当的注射器直径为您正在使用的注射器大小（见注射器泵手册的指引）。 </li><li>设置电磁炉〜38 ° C和覆盖用纸巾或其他薄膜屏障，以保护幼崽，在恢复阶段。 </li></ol> 3。注射程序和小狗恢复<ol><li>通过对他们进行冷麻醉IACUC批准协议后，准备注射P0或P1鼠标幼仔。挫伤一些纸巾与水和冰的地方。广场上的纸巾（保持他们良好的分离）的幼崽所需的数字（4-5），并轻轻地折叠，使他们对幼崽的纸巾湿纸巾覆盖。轻轻地放置纸巾上的少量碎冰和孵化幼崽，大约5分钟。幼崽可以保持长达15分钟的上冰，收回罚款事后。 </li><li>后小狗是足够的麻醉，放置在一个固定块（我们使用的泡沫塑料块，通常持有15毫升管）和注射部位的最佳途径，它的位置。皮层，它是最简单的方法，奠定其腹动物的单位，而小脑是很有帮助的小狗的头块的边缘位置，以提供更好的访问的头骨后部。小狗可能被固定在一个创可贴，如果需要的话。 </li><li>一方面小狗的头，而​​用另一只手在所需的站点通过插入头骨针手动到位。它是有帮助的拉扯皮肤紧，以防止注射过程中移动。皮层，它是有益的，使一个明确的解剖标志，如颅骨的lambda缝合，注射，因为这将有助于在未来的注射。通过头骨小脑可以看出，作为一个组织直接躺在尾鳍的丘细条状。应插入针深度略有不同动物个体之间和株，可以通过针预定标记衡量，但皮质和小脑，我们发现，从表面的深度〜0.5 - 1毫米通常适合。其他网站的插入深度应确定实验的最终用户。 注:针应该轻易地穿透颅骨，如果它不，不推太难，因为这会伤害动物。重新定位针，轻轻再试一次。 </li><li>注入病毒解决方案。该注射液是开始由按泵上的START键，或令人沮丧的脚踏板，根据模型的压力注射器使用。在一个脚踏板的情况下，建议有一个同事协助按下按钮，以尽量减少不必要的手部动作，可能会影响注射。 </li><li>选定的卷已被注射后，保持在针几秒钟，然后轻轻滑动顺利取出针。务必抓住小狗的头，以尽量减少多余的动作。由于注射设备与乙醇的实验之间的清洗和消毒伤口小，使用防腐剂是不必要的。 </li><li>放在有盖的电磁炉与温度设定在38〜小狗° C到温暖的恢复，通常5-10分钟。在此期间，您可能会继续注入其他幼仔。 </li><li>所有的幼崽后已痊愈（他们应该是粉红色和移动）的家庭床上用品的一部分，并轻轻揉搓它的幼仔。返回的幼鼠的母亲为一组。 </li></ol>注:这是非常重要，以确保幼仔温暖和暴露在家里床上用品，返回他们的母亲和作为一个群体，他们返回之前。如果不这样做可能会导致伤害或杀死幼崽的母亲。 4。设备清洗<ol><li>注射后是完整的和幼崽已返回他们的母亲，清洁，完全用丙酮或70％的乙醇加载注射器注射设置。通过注射几次安装的解决方案，每次使用新鲜的溶液冲洗。这将删除任何剩余的病毒解决方案，并沉淀。丙酮或乙醇，然后蒸发。 注:丙酮是首选的清洁，但如果你的安装使用任何丙酮敏感元件，如氰基丙烯酸酯粘合剂，然后用70％的乙醇对这些步骤。 </li><li>消毒漂白水和丢弃废物的工作区，在一个适当的生物危害容器中。 </li></ol> 5。分析<ol><li>牺牲IACUC批准的安乐死方法所需年龄的动物。灌注固定，建议在以后的感染，特别是在成年动物神经元，可视援助。 </li><li>标准的免疫程序，可用于荧光信号放大。简言之，100微米浮动vibratome部分，与PBS +1％Triton X - 100的的透和PBS +1％Triton X - 100的的10％正常山羊血清阻止。第孵育阻断抗体在4℃过夜，额外的清洗和阻塞的解决方案。部分，然后培养在阻塞的解决方案与二级抗体在室温下2小时，彻底洗净，在幻灯片上安装防褪色的安装媒体coverslipped。冰冻切片也可用于可视化标记的神经元，但内源性荧光可能会丢失。 </li></ol> 6。代表性的成果: 一个成功的注射过程会产生强大的感染地区没有观察到的组织损伤或其他不良影响的动物在注射部位的神经元。如图1所示，可以观察到感染的蔓延，在创建记者小鼠（ROSA26R）rAAV8 - Cre重组注入不同解剖部位的12。到特定的地区，如皮层和上丘注射，通常产生并蔓延到最小邻近地区（图1A - B）的局部感染。高滴度的病毒解决方案的使用，使感染邻近地区，如海马，更容易（图1A）。注射用低滴度病毒感染稀疏解决的结果，通常保持注射部位附近，rAAV8 - Cre重组小脑注​​射ROSA26R记者小鼠（图1C - D）中线。我们观察ROSA26R记者小鼠重组注射rAAV8 - Cre重组后2天之内，建议，都可以发生感染，重组和报告基因的表达较快。此外，我们已经成功地使用这种技术研究在非 - 罗萨使用floxed有条件的等位基因位点基因缺失的影响，其结果将刊登在别处。 感染细胞的数量应与注入的病毒解决方案的滴度相关。例如，注射的两个rAAV8到小脑表达不同荧光蛋白的病毒的高滴度解决方案的混合感染浦肯野许多差异标记的细胞（图2A）。相反，注射低滴度病毒解决方案，可以在稀疏感染援助在可视化的单细胞（图2B）的结果。可直接观察到荧光标记的神经元在鲜切花，活组织（图2B），或可以受到免疫宽视场或合作，以提高以后的图像采集的内源性荧光信号nfocal荧光显微镜（图2A，图3A - C）。最后，rAAV8皮层与精细的工序强大的标签（图3A - C），在感染的神经元类型的各种能力。 <img alt="图1" src="/files/ftp_upload/2823/2823fig1.jpg" /> 图1:LacZ的ROSA26R记者在P0 rAAV8 - Cre重组注入老鼠的大脑中的染色表明Cre重组活动和感染的位置。 <ol type="A"><li>宽领域的矢状查看了P14的大脑呈现出高滴度（1 × 10 12 GC /毫升）注射到皮层和上丘。使用高滴度的病毒，使得它更可能是邻近地区也会被感染。 </li><li> P14的皮质矢状切面注射低滴度（1 × 10 11 GC /毫升），少传播病毒的演示与本地感染。 </li><li>注射低滴度的中线（1 × 10 9 GC /毫升）病毒解决方案的一个P28小脑wholemount的观点表明，感染是稀疏的，和通常的注射部位附近。 </li><li>在C小脑中线的矢状查看）。请注意，一般只Purkinje细胞rAAV8感染。 </li></ol><img alt="图2" src="/files/ftp_upload/2823/2823fig2.jpg" /> 图2:rAAV8表达荧光蛋白的高与低滴度注射小脑标签 。 <ol type="A"><li>矢状切面的P21cerebellum两个病毒表达EGFP（绿色）和DsRed的（红色）的混合感染。高滴度的病毒混合感染的浦肯野细胞的大量。 </li><li>一个在P14的生活非​​常低滴度病毒感染小脑浦肯野细胞成像后立即清扫。 </li></ol><img alt="图3" src="/files/ftp_upload/2823/2823fig3.jpg" /> 图3:rAAV8 - DsRed的皮质标签。 各种皮层神经元类型的例子，在一个P21的皮层在P0 rAAV8 - DsRed的，是由感染冠状切片。

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没有利益冲突的声明。

这里介绍的新生儿病毒注射方法提供了一个简单而快速的方式来产生体内马赛克产后大脑发育的研究。该方法需要利用floxed等位基因目前可用的，以及那些正在通过高通量基因靶向项目6。使用Cre转基因表达相比，这种方法提供了一种快速的方法来测试基因在不同细胞类型的功能，携带floxed等位基因的小鼠可直接用于实验，整个病毒注射过程从开始到结束，可在不到一小时完成。此?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> rAAV8 - CRE，- GFP - DsRed的， </td><td>矢量Biolabs公司</td><td> ＃7060，＃7061，客户订单</td><td> http://www.vectorbiolabs.com </td></tr><tr><td>哈佛泵11加</td><td>哈佛器械</td><td> ＃702208 </td><td> （没有脚踏板端口） </td></tr><tr><td>视网膜色素上皮细胞注射套件</td><td>世界精密仪器</td><td> RPE - KIT </td><td>包含连通管，注射针（36G），持针器</td></tr><tr><td> NanoFil注射器，100微升</td><td>世界精密仪器</td><td> NANOFIL - 100 </td><td>包括可重复使用的装载针</td></tr><tr><td> D - PBS </td><td> Invitrogen公司</td><td> 14040-117 </td><td></td></tr><tr><td> GFP抗体</td><td>鸟类</td><td> GFP - 1020 </td><td></td></tr><tr><td> DsRed的抗体</td><td> Clontech公司</td><td> 632496 </td><td></td></tr><tr><td>加热块</td><td> VWR </td><td> 97042-610 </td><td></td></tr><tr><td> ROSA26R鼠标</td><td>杰克逊实验室</td><td> 003309 </td><td></td></tr></tbody></table>

mosaic analysis of gene function in postnatal mouse brain development by using virus-based cre recombination

不仅依赖于胚胎发育的主要神经通路建立正常的脑功能，但也发育时神经回路的成熟和完善。 Misregulation在这个阶段，可能会导致神经和精神疾病，如自闭症和精神分裂症1,2。许多基因已在胎儿的大脑研究，发现许多 3-5发育过程至关重要。然而，他们在出生后大脑功能在很大程度上是未知的，部分原因是因为他们在小鼠的缺失往往会导致新生儿发育期间，以杀伤力，部分原因是因为他们在早期发展的要求，阻碍了产后的分析。为了克服这些障碍，这些基因的floxed等位基因，目前正在生成在小鼠 6 。当结合表达Cre重组酶在特定的细胞类型，有条件的删除，可以实现在产后的大脑研究基因功能的转基因等位基因。然而，这种方法需要额外的等位基因和额外的时间（3-6个月），以产生适当的基因型的小鼠，从而限制了在鼠脑大规模扩展的遗传分析。 在这里，我们展示了一个互补的方法，使用病毒的表达Cre重组迅速，系统地研究这些floxed等位基因在出生后大脑发育。通过注入重组腺相关病毒（rAAVs）7,8编码的Cre新生儿的大脑，我们可以删除该基因在大脑的不同区域的利益。通过控制病毒滴度和共表达荧光蛋白标记，我们可以同时实现镶嵌基因的失活和稀疏神经元的标签。这种方法绕过了许多基因在早期发展的要求，使我们能够学习他们的细胞自主的功能在许多关键的过程，在出生后大脑发育，包括轴突和树突状增长，分支，和平铺，以及突触的形成和完善。该方法已成功地使用在我们自己的实验室（未公布结果）和其他8,9，并可以扩展到其他的病毒， 如 9，以及慢病毒的shRNA或主导活性蛋白 10的表达，。此外，通过与电相结合的技术，以及最近开发的光学成像工具11，这种方法提供了一个新的战略，以研究如何遗传途径影响神经电路的发展和在小鼠和大鼠的功能。

Watch this Scientific Journal Video about Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination at JoVE.com

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

一个<em&gt;在体内</em&gt;产后脑的方法来测试基因功能描述。表达CRE和/或荧光蛋白的重组自动增值服务注入新生小鼠大脑。马赛克基因的失活和稀疏的神经标签实现，使神经电路的发展过程的关键基因功能的快速分析。

利用基于病毒的Cre重组马赛克产后小鼠脑开发基因功能分析

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

mosaic-analysis-gene-function-postnatal-mouse-brain-development-using

Research

JoVE Journal

Neuroscience

11.9K 次观看.  Keck School of Medicine, University of Southern California. 一个在体内产后脑的方法来测试基因功能描述。表达CRE和/或荧光蛋白的重组自动增值服务注入新生小鼠大脑。马赛克基因的失活和稀疏的神经标签实现，使神经电路的发展过程的关键基因功能的快速分析。

视频: Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

基因传递到产后大鼠脑非心室质粒注射和电穿孔

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

科研

神经科学

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔:鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Interneuron 前体 Homochronic 移植早期小鼠脑的研究

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

脑磁图在儿童脑功能研究中的应用

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

与腺相关病毒的向量的生产、纯化和质量控制

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

常见马莫塞的全皮质电皮质阵列的慢性植入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Methods Article

利用基于病毒的Cre重组马赛克产后小鼠脑开发基因功能分析