Booth Jonathan R H, Sane Varun, Gather Malte C, Pulver Stefan R
SUPA, School of Physics and Astronomy, University of St Andrews, St Andrews, United Kingdom.
School of Psychology and Neuroscience, University of St Andrews, St Andrews, United Kingdom.
J Undergrad Neurosci Educ. 2020 Dec 31;19(1):A124-A133. eCollection 2020 Fall.
Central pattern generators (CPGs) are neural networks that produce rhythmic motor activity in the absence of sensory input. CPGs produce 'fictive' behaviours which parallel activity seen in intact animals. CPG networks have been identified in a wide variety of model organisms and have been shown to be critical for generating rhythmic behaviours such as swimming, walking, chewing and breathing. Work with CPG preparations has led to fundamental advances in neuroscience; however, most CPG preparations involve intensive dissections and require sophisticated electrophysiology equipment, making export to teaching laboratories problematic. Here we present an integrated approach for bringing the study of locomotor CPGs in larvae into teaching laboratories. First, we present freely available genetic constructs that enable educators to express genetically encoded calcium indicators in cells of interest in the larval central nervous system. Next, we describe how to isolate the larval central nervous system and prepare it for live imaging. We then show how to modify standard compound microscopes to enable fluorescent imaging using 3D printed materials and inexpensive optical components. Finally, we show how to use the free image analysis programme ImageJ and freely available features in the signal analysis programme DataView to analyse rhythmic CPG activity in the larval CNS. Comparison of results to those obtained on research equipment shows that signal-to-noise levels are comparable and core features of larval CPG activity can be observed. Overall, this work shows the viability of exporting live imaging experiments to low cost environments and paves the way for new teaching laboratory exercises revolving around optical imaging of CPG activity.
中枢模式发生器（CPGs）是在没有感觉输入的情况下产生节律性运动活动的神经网络。CPGs产生“虚构”行为，这些行为与完整动物中观察到的活动相似。CPG网络已在多种模式生物中被识别，并已被证明对于产生诸如游泳、行走、咀嚼和呼吸等节律性行为至关重要。对CPG制剂的研究已在神经科学领域取得了根本性进展；然而，大多数CPG制剂需要密集解剖且需要复杂的电生理设备，这使得向教学实验室推广存在问题。在此，我们提出一种将幼虫运动CPGs的研究引入教学实验室的综合方法。首先，我们提供免费的基因构建体，使教育工作者能够在幼虫中枢神经系统的感兴趣细胞中表达基因编码的钙指示剂。接下来，我们描述如何分离幼虫中枢神经系统并为活体成像做准备。然后，我们展示如何使用3D打印材料和廉价的光学元件对标准复合显微镜进行改造以实现荧光成像。最后，我们展示如何使用免费的图像分析程序ImageJ和信号分析程序DataView中的免费功能来分析幼虫中枢神经系统中有节律的CPG活动。将结果与在研究设备上获得的结果进行比较表明，信噪比水平相当，并且可以观察到幼虫CPG活动的核心特征。总体而言，这项工作展示了将活体成像实验推广到低成本环境的可行性，并为围绕CPG活动光学成像的新教学实验室练习铺平了道路。
