Moreno Rosa L, Josey Megan, Ribera Angeles B
Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus (UCAMC);
Neuroscience Graduate Program, University of Colorado Anschutz Medical Campus (UCAMC).
J Vis Exp. 2017 Apr 18(122):55507. doi: 10.3791/55507.
Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model. For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages.
斑马鱼最初作为一种发育模型被引入，如今在许多其他领域也颇受欢迎。能够轻松饲养大量快速发育的生物体，再加上胚胎具有光学透明性，这些最初成为了该模型极具吸引力的特性。在过去的二十年里，这种模型的成功因易于进行大规模诱变筛选以及转基因操作简便而得到进一步推动。最近，基因编辑方法更是增强了该模型的功能。对于神经发育研究而言，斑马鱼胚胎和幼体提供了一个可以应用多种方法的模型。在此，我们聚焦于能够研究神经元一项基本特性——电兴奋性的方法。我们对斑马鱼脊髓神经元进行电生理研究的准备工作包括使用兽用缝合胶将标本固定在记录室中。从斑马鱼胚胎和幼体进行记录的其他方法包括使用细钨针将标本固定在记录室上。钨针最常用于将标本以侧向方式固定，不过也曾用于将幼体背侧向上固定。缝合胶已被用于以两种方向固定胚胎和幼体。使用这种胶水，可以进行最小限度的解剖，无需酶处理就能接触到脊髓神经元，从而避免任何由此产生的损伤。然而，对于幼体，有必要进行短暂的酶处理以去除脊髓周围的肌肉组织。这里描述的方法已被用于研究几个发育阶段的运动神经元、中间神经元和感觉神经元的内在电特性。
