作者要感谢鲍勃·弗里曼为培训和克氏针实验室的使用的青蛙设施，凡向量子实验室的支持。我们感谢哈佛医学院的尼康影像中心寻求帮助，在光学显微镜图1中的图像。这项工作是由以下内容：NRSA美国国立卫生研究院的奖学金和NIH K99奖学金拉尔，基础科学伙伴关系的资金（ <a href="https://bsp.med.harvard.edu/" target="_blank">https://bsp.med.harvard.edu/</a> ）AEF和NIH RO1 NS035909 DVV

注：我们描述的前两部分只在简单的步骤，优良的协议的详细信息已在其他地方发表，重点更具体步骤（ 如 8-12）。此外，一般的工作与非洲爪蟾脊髓神经元在活细胞培养的协议已经发表在了详细的方法第8条。我们强烈建议审查该文章的这个视频作​​为一种补充，虽然在这里我们提供足够的信息来成功地执行和故障诊断神经管的解剖和电镀协议在第3步中。 1。 非洲爪蟾卵的体外受精<ol><li>获得卵雌蛙注射绒毛膜促性腺激素（400单位/青蛙）12-18小时前集蛋。收集鸡蛋1X马克的修改林格氏液（MMR）（0.1 M氯化钠，2.0毫米2.0毫米，1.0毫米硫酸镁 ，氯化钾氯化钙，5.0毫摩尔HEPES，pH值7.4 9）。 </li><li>施肥鸡蛋在体外用剁碎睾丸，如前所述9。 </li><li>至少20分钟后，取出胚胎果冻外套在2％以上的半胱氨酸在1X MMR（带来了用NaOH至pH为7.8）为3-5分钟孵育胚胎。洗净0.1X MMR（或0.1％的MBS（修改巴特的盐水，1X配方：88毫米氯化钠，氯化钾1毫米，0.7毫米氯化钙 ，1毫米硫酸镁 ， 碳酸氢钠 2.5毫米，5毫米的HEPES，pH值7.8））3 -5倍，并保持在室温下的胚胎，直到注射，或如果需要的话，地方胚胎在14-18°C至发展缓慢。 </li></ol> 2。微量注射RNA 注意：虽然我们在这里利用RNA注射，这些技术并不限于核糖核酸，并也可用于DNA，蛋白质，或修饰的核酸的基因操作和以前已经描述了12。 <ol><li>在实验中，RNA的标记细胞骨架或其他结构ARË使用mMessage mMachine试剂盒（Ambion公司）的线性DNA模板的转录。需要的mRNA的5&#39;帽和一个3&#39;多聚腺苷酸尾促进翻译和防止退化在胚胎。 PCS2 +是一种常用的用于此目的的载体，和转录试剂盒包括一个帽类似物的合成反应过程中。我们重新悬浮于无核酸酶的DDH 2 0的转录的mRNA，在500和2000之间的库存浓度纳克/微升，然后按提示存储于-80℃。在注射前，稀DDH 2 O中的RNA注射到适当的浓度。因为只有一小体积将被注入胚胎，再悬浮在缓冲液中是没有必要的。我们的最终注入浓度通常是50-200皮克/ NL，根据该构造，我们通常将注入每个胚胎至4 NL，尽管传统上，许多实验室注入10 NL，这是总体积的约1％的一个胚胎。 RNA可以是在高剂量的毒性，胚胎属的剂量为每从10pg LLY取值范围为1 ng，虽然可以耐受高达5纳克取决于特定的基因和纯度。然而，对于成像蛋白质的定位，应尽可能低的水平注入，以避免过度表达的文物。 RNA EB1-GFP在本协议中的最终金额为250微克每胎。 </li><li>准备通过拉动针拉出器9，毛细管的前端直径为〜0.2μm的注射针头。随着萨特P-87拉拔器，我们使用下面的设置：热644，拉125，速度70，250，但这些参数的不同而不同的机器上，必须重新调整，改变后的灯丝（我们目前使用的2.5毫米方盒子丝，宽度为2.5 mm）。在显微镜下，用钳子打破针尖以一个角度到生成羽毛笔状。还有其它的方法为打破和填充注射针（例如，11,12），但我们通过将一小滴（0.5 - 1微升）的后端与RNA填充针针。对于显微注射到胚胎，有一些市售的喷射系统，两个最常见的是的Pico-喷射器（医疗系统），和Nanoject（德拉蒙德科学）（参见图11的详细信息）。我们使用的医疗系统PLI-100的Pico-喷油器，它使用的数字设定的时间内，以提供一致的纳升体积的压缩气体。注射体积必须被校准为每个新的微量吸移管，使用一个阶段微米，和注射时间应进行调整，以达到所需量的注射核糖核酸。 </li><li>地点受精胚胎在1 - 4 - 细胞阶段的塑料培养皿含有5％聚蔗糖的0.1倍MMR。用钳子到位握住胚胎，或放置在一个保持平台（塑料网，与一个〜1毫米的网格中，附着于底部的菜用橡皮泥或牙科用蜡），注入到动物卵裂球所需音量（参见步骤2.1进样体积）的详细信息。分布在几个禄ations整个胚胎，所述至少一个喷射到每个动物卵裂球，以获得更均匀的分布（例如，一个4 - 细胞阶段的胚胎中，我们通常注入在每个卵裂球的1-2倍，而在2 - 细胞阶段胚胎中，我们注入的2-4倍，在每一个卵裂球）。等待2-4细胞阶段的一个好处是，你保证胚胎正常开始切割。但是，如果时间是最重要的，你可以在1 - 细胞期开始，在最终的结果，除需要注入更多的胚胎差别很小。可以使用旧的阶段的胚胎，这是特别有用的，如果特定的细胞类型有针对性的注射，但我们一般喜欢更广阔的表达，我们在年轻的胚胎注入更多的多次注资，以减少需要。 </li><li>注射的胚胎转移到一个塑料培养皿中0.1X MMR，并让他们发展到阶段20日至23日13。孵育胚胎在14至22°C，根据所需的SPEED的发展。 （解剖神经管次日，使用〜22°C的一天后，使用〜14°C） </li></ol> 3。神经管的解剖和电镀<ol><li>在此之前进行解剖，准备培养皿14。首先，外套盖玻片池的表面上方的盖玻片上，Mattek或的Lab-Tek培养皿，并孵育一小时（或者200微克/毫升多聚赖氨酸在PBS中有足够的溶液，人们可以使用玻璃盖玻片坐在宽松的培养皿中，后放置在载玻片上的成像和免疫组化）。吸干，并用PBS超过3次，让干。然后，用10μg/ ml的层粘连蛋白在PBS中的外套菜肴（我们通常使用500微升每盖玻片，足以在表面上池）一小时，在37度。吸液层粘连蛋白的溶液，并用过量的PBS洗3次，小心不要让层粘连蛋白涂层的表面成为暴露到空中接口。更换ë，使用培养基的PBS（50％林格氏液，49％的L-15的媒体，1％胎牛血清，加50微克/ ml青霉素/链霉素和庆大霉素，pH值7.4，过滤灭菌）。 （林格氏媒体，115 mM氯化钠，氯化钾2.5毫米，2毫米氯化钙 ，10 mM HEPES的pH为7.4 mM的EDTA，0.5）。虽然我们的协议，使用这个媒体，应该指出的是，这是一个丰富 ​​的媒体共同为老年人爪蟾视网膜神经节培养的神经元减少能源保留。年轻的脊髓文化的生存和成长为超过24小时的纯林格的媒体，无需额​​外的增长因素，这确实是使用该系统的好处之一。可选：添加NT3和​​BDNF（最终浓度为25纳克/微升）培养基中增加轴突生长。 </li><li>由于注入的mRNA的表达的变异，胚胎可能表现出的荧光的马赛克。在此之前执行解剖，屏幕胚胎荧光的存在下，以确定这些胚胎EXPRESS的荧光融合蛋白在神经管。将荧光胚胎阶段20-23到琼脂糖Steinberg的媒体（58毫摩尔NaCl，0.67 mM KCl中，0.44毫摩尔的Ca（NO 3）2，1.3 mM的用MgSO 4，4.6毫摩尔Tris，pH值至7.8，则充满了涂覆的塑料培养皿蒸压）5。为了使琼脂糖凝胶涂层菜，外套塑料盘底部的融化的1％琼脂糖0.1X MMR，让硬化。这提供了一个柔软的表面上，所以，不会发生损坏的罚款钳和钨针，在随后的解剖步骤。 23超越阶段的胚胎解剖是可能的，但它是更具挑战性的组织坚持更加紧密地彼此，因此需要较长的治疗与胶原酶。 </li><li>在解剖的范围，删除用细镊子，卵黄膜，然后隔离整个背侧部的胚胎，进行了一系列的切口。当一个镊子胚胎在地方，使用第二个，做一个切口侧的胚胎暴露中空的内部。然后，用两个钳子捏沿背，腹部分的胚胎组织之间，从而削减一半，分离胚胎神经管的背侧部。 </li><li>为15-20分钟，在旋转器上，松开组织，然后：移液器，背侧外植体到与新鲜Steinberg的溶液琼脂糖涂层的塑料培养皿中，将背侧的外植体在Eppendorf管中的2毫克/毫升胶原酶在Steinberg的媒体中。可选择地，外植体可以被放置在培养皿含有胶原酶溶液15分钟后，在整个剥离，然后可以进行。 </li><li>使用钳子，轻轻地剖析了神经管背表皮和腹脊索。滑动的前端之间的表皮和皮下组织的钳子，慢慢拉回来的表皮，揭示神经管的下方。然后，使用1钳子组织保持和另一个以滑动的尖端之间的神经管和脊索。最后，使用镊子去除任一侧上的管的体节。 </li><li>神经管转移到装满培养基琼脂糖涂覆培养皿，在步骤3.1中所描述。 </li><li>几个神经管收集后，切成无数颗（约20是孤立的，如果整个管）磨钨针或镊子，然后在培养皿板件充满介质（步骤3.1编制），传播外植体均匀地行。 </li><li>电镀后，不动​​的菜，因为这会干扰安装的细胞。镀神经管外植体孵育约20-22°C。我们通常的菜留在板凳上的细胞在室温下过夜。 非洲爪蟾神经元的好处之一是，无需特殊的培养箱中培养细胞培养，调节CO 2水平或温度。神经突起生长锥，可以观察和成像，在室温12-24小时后，化学镀，虽然生长可以加快与添加的生长因子。在一般情况下，表达的RNA或质粒的产品会导致在变量细胞之间的表达，并因此可能存在的范围内的荧光之间的表达水平生长锥。我们已观察到电镀后持续至48小时以外的荧光蛋白的RNA的表达，但是这依赖于特定的结构。 </li></ol> 4。代表性的成果继后的协议，该协议被概括在图1A中，将发送健康，正确的解剖和培养的神经外植体在层粘连蛋白/多聚赖氨酸的基板， 如图1B中所示的每一个方向上的众多轴突或神经突。提示的轴突的生长锥，无论是DIC的光学元件，在图1C，整体活力动态图像，或高分辨率荧光显微镜检查可成像的LOcalization荧光标记的细胞骨架蛋白，例如，EB1-GFP 如图1D所示。 <img alt="图1" src="/files/ftp_upload/4232/4232fig1.jpg" /> 图1总结的实验方案和预期的结果。 A）协议的流程图。在第3天的解剖细节，请参阅电影。整个神经管应切成约20个大小相等的块，并均匀地分布在盖玻片上的行展开。在这幅漫画描绘为代表，剪刀，切割组织用细镊子。 CG绒毛膜促性腺激素B）DIC图像的轴突或神经突起生长出来的外植体植（在左上角）。 C）放大图像的不断增长的轴突生长锥尖。 D）荧光显微镜图像的EB1-GFP在生长锥。比例尺10微米。 <a href="http://www.jove.com/files/ftp_upload/4232/4232fig1large.jpg" target="_blank">点击此处查看大图</a> 。

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没有利益冲突的声明。

非洲爪蟾神经外植体发出神经突起，在一个非常强大的方式24小时后电镀层粘连蛋白/聚赖氨酸基板上，如果条件合适的。该基板上，生长锥高度运动，并可以实现轴突长度可达1毫米，在所有方向上从外植体向外延伸，虽然典型的长度为100微米或以上。如果神经突起不生长出来，有这种情况的原因是这种情况的数量是有限的。一种可能性是，神经外植体没有正确坚持的菜。确保不破坏附件?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>绒毛膜促性腺激素</td><td>银色化学实验室</td><td> C-HCG-ON-10 </td></tr><tr><td>半胱氨酸</td><td> Sigma-Aldrich公司</td><td> 52-90-4 </td></tr><tr><td> mMessage mMachine试剂盒</td><td> Ambion公司</td><td> AM1340 </td></tr><tr><td>毛细管Borosil针1.2毫米（外径）x 0.9毫米（ID） </td><td> FHC </td><td> 30-31-0 </td></tr><tr><td> FICOLL </td><td>西格玛</td><td> F2637 </td></tr><tr><td>杜蒙第5 Biologie Inox公司钳</td><td>精细科学工具</td><td> 11252-20 </td></tr><tr><td>胶原酶</td><td> Sigma-Aldrich公司</td><td> 9001-12-1 </td></tr><tr><td> Mattek菜</td><td>垫泰克公司</td><td> P35G-1.5-14-C </td></tr><tr><td> L-15的</td><td> Invitrogen公司</td><td> 21083-027 </td></tr><tr><td>聚-L-LYSI东北</td><td>西格玛</td><td> P-1399 </td></tr><tr><td>层粘连蛋白</td><td>西格玛</td><td> L2020 </td></tr><tr><td> NT3 </td><td>西格玛</td><td> N1905 </td></tr><tr><td> BDNF </td><td>西格玛</td><td> B3795 </td></tr></tbody></table>

neural explant cultures from xenopus laevis

轴突引导的复杂过程，主要是由，这是动态能动结构的尖端处的生长的轴突生长锥。在轴突生长，生长锥必须整合多种来源的指导提示信息，调节细胞的骨架，以推动生长锥和准确定位找到它的具体目标1。这种整合发生在细胞骨架的水平如何，仍是新兴的，在生长锥细胞骨架蛋白和效应动力学检查可以让这些机制的阐明。 非洲爪蟾生长锥足够大（直径10-30微米）进行高的高分辨率实时成像细胞骨架的动态（ 如 2-4），很容易分离和操作在实验室设置比其他脊椎动物。青蛙是一个典型的模型系统发育神经生物学研究，以及重要的早期洞察生长锥话筒最初发现使用该系统5-7 rotubule动态。在此方法中8，鸡蛋收集受精卵在体外 ，注入与RNA编码荧光标记的细胞骨架的融合蛋白或其他结构，操纵基因的表达，然后使其发展到神经管阶段。神经管分离解剖，然后进行培养，和不敷神经突起生长锥成像。在这篇文章中，我们将介绍如何执行此方法，我们的目标，就是要培养非洲爪蟾生长锥为后续的高清晰度图像分析。虽然我们提供+ TIP融合蛋白EB1-GFP的例子中，此方法可应用于任意数量的蛋白质生长锥内澄清他们的行为。

Watch this Scientific Journal Video about Neural Explant Cultures from Xenopus laevis at JoVE.com

Neural Explant Cultures from Xenopus laevis

培养神经外植体解剖<em&gt;非洲爪蟾</em表达荧光融合蛋白的胚胎，使成像的生长锥细胞骨架的动态。

的神经植体培养<em&gt;非洲爪蟾</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

neural-explant-cultures-from-xenopus-laevis

Research

JoVE Journal

Neuroscience

Neural Explant Cultures from Xenopus laevis

12.4K 次观看.  Harvard Medical School. 培养神经外植体解剖非洲爪蟾

视频: Neural Explant Cultures from Xenopus laevis

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

科研

神经科学

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

同时前和突触后电生理记录<em&gt;爪蟾</em神经肌肉文化

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

一个简单的行为分析在检测视觉功能<em&gt;非洲爪蟾</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

从尸检人脑软脑膜捐助外植体培养的推导

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...