| Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells
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Author:
Date:
2017-09-05
[Abstract] Cardiac neural crest cells (CNCCs) originate at the dorsal edge of the neural tube between the otic pit and the caudal edge of the 3rd somite, and migrate into the pharyngeal arches and the heart. We have shown that fibronectin (Fn1) plays an important role in the development of the CNCC by regulating the differentiation of CNCCs into vascular smooth muscle cells around pharyngeal arch arteries (Wang and Astrof, 2016). This protocol describes the isolation of CNCCs from the neural tube and from the caudal pharyngeal arches, and the differentiation of neural crest-derived cells into smooth muscle cells. This protocol was adapted from (Newgreen and Murphy, 2000; Pfaltzgraff et al., 2012).
[摘要] 心脏神经嵴细胞（CNCC）起源于神经管的背部边缘，位于第3个体节的耳穴和尾缘之间，并迁移到咽弓和心脏。 我们已经表明，纤连蛋白（Fn1）通过调节CNCCs到咽弓动脉周围的血管平滑肌细胞的分化，在CNCC的发展中起重要作用（Wang and Astrof，2016）。 该方案描述了CNCC与神经管和尾尾弓的分离，以及神经嵴衍生细胞分化成平滑肌细胞。 该方案从（Newgreen和Murphy，2000; Pfaltzgraff等人，2012）改编。
【背景】以前发表的方案描述了从神经管分离神经嵴细胞。然而，在耳孔和第三体细胞之间的神经管区域中的神经嵴细胞包括有助于许多不同细胞类型的神经嵴细胞群体;例如，迷走神经嵴细胞也来自该区域。在该方案中，我们修改了用于分离心脏神经嵴细胞的常规方法。而不是使用神经管，我们在胚胎期（E）9.5（22-25个体节期）使用尾部咽部弓形区。这是在将心脏神经嵴细胞分化为血管平滑肌细胞之前。神经嵴培养物通常含有污染性间充质细胞，通常表达平滑肌基因。为了鉴定神经嵴衍生细胞，我们从以下交叉产生的胚胎中分离出神经嵴细胞：Fn1flox / flox; ROSAmTmG / mTmG雌性小鼠×Fn1 +/-;Tfap2αIRESCre/ ...
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| Spinal Cord Preparation from Adult Red-eared Turtles for Electrophysiological Recordings during Motor Activity
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Author:
Date:
2017-07-05
[Abstract] Although it is known that the generation of movements is performed to a large extent in neuronal circuits located in the spinal cord, the involved mechanisms are still unclear. The turtle as a model system for investigating spinal motor activity has advantages, which far exceeds those of model systems using other animals. The high resistance to anoxia allows for investigation of the fully developed and adult spinal circuitry, as opposed to mammals, which are sensitive to anoxia and where using neonates are often required to remedy the problems. The turtle is mechanically stable and natural sensory inputs can induce multiple complex motor behaviors, without the need for application of neurochemicals. Here, we provide a detailed protocol of how to make the adult turtle preparation, also ...
[摘要] 虽然已知在位于脊髓的神经元回路中很大程度地进行运动的产生，但是所涉及的机制仍不清楚。乌龟作为调查脊柱运动活动的示范系统具有优势，远远超过使用其他动物的模型系统。对缺氧的高抗性允许对完全发育和成年脊髓电路进行调查，而不是对缺氧敏感的哺乳动物，并且通常需要使用新生儿来补救问题。乌龟是机械稳定的，天然感觉输入可以诱导多种复杂的运动行为，而不需要神经化学物质的应用。在这里，我们提供了如何使成年龟准备的详细方案，也称为电生理调查的综合准备。在这里，通过机械感觉激活，通过细胞内，细胞外和电图记录来记录单细胞和网络活性，可以诱导后肢刮擦反射。该准备工作是由Petersen等人（2014）和Petersen和Berg（2016）以及其他正在进行的研究开发的。
【背景】脊髓电生理学的研究传统上与机械并发症有关，因为许多运动部件和脊柱的灵活性。为了规避这个问题，脊髓经常从柱中解剖出来并移动到可以进行稳定的电生理记录的室。然而，该过程具有缺点，例如，如果要研究多个电动机行为，则适当激活电动机电路是复杂的。此外，缺乏供血和缺乏氧气对电路的健康和完整性有严重影响。一个可以规避所有这些问题的实验模型是龟制剂（Keifer and ...
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| Neurite Outgrowth Assay
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Author:
Date:
2016-01-05
[Abstract] Neurite outgrowth in culture provides an easy way to determine the effects of a particular substrate or exogenous factor on neuron behavior. Dissociated neurons can be plated on a variety of substrates and the length of the longest neurite outgrowth can be compared. Here, we describe how to isolate and dissociate dorsal root ganglion (DRG) neurons, culture them on coverslips, and measure longest neurite outgrowth.
[摘要] 文化中的神经细胞生长提供了一种简单的方法来确定特定底物或外源因子对神经元行为的影响。 分离的神经元可以铺在各种底物上，并且可以比较最长的神经突生长的长度。 在这里，我们描述如何分离和解离背根神经节（DRG）神经元，将其培养在盖玻片上，并测量最长的神经突生长。
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