Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, USA.
Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Nat Protoc. 2019 May;14(5):1455-1488. doi: 10.1038/s41596-019-0146-6. Epub 2019 Apr 5.
Sleep is nearly universal among animals, yet remains poorly understood. Recent work has leveraged simple model organisms, such as Caenorhabditis elegans and Drosophila melanogaster larvae, to investigate the genetic and neural bases of sleep. However, manual methods of recording sleep behavior in these systems are labor intensive and low in throughput. To address these limitations, we developed methods for quantitative imaging of individual animals cultivated in custom microfabricated multiwell substrates, and used them to elucidate molecular mechanisms underlying sleep. Here, we describe the steps necessary to design, produce, and image these plates, as well as analyze the resulting behavioral data. We also describe approaches for experimentally manipulating sleep. Following these procedures, after ~2 h of experimental preparation, we are able to simultaneously image 24 C. elegans from the second larval stage to adult stages or 20 Drosophila larvae during the second instar life stage at a spatial resolution of 10 or 27 µm, respectively. Although this system has been optimized to measure activity and quiescence in Caenorhabditis larvae and adults and in Drosophila larvae, it can also be used to assess other behaviors over short or long periods. Moreover, with minor modifications, it can be adapted for the behavioral monitoring of a wide range of small animals.
睡眠在动物中几乎普遍存在，但仍未被充分理解。最近的工作利用简单的模式生物，如秀丽隐杆线虫和黑腹果蝇幼虫，来研究睡眠的遗传和神经基础。然而，在这些系统中手动记录睡眠行为既费力又低效率。为了解决这些限制，我们开发了用于在定制微加工多孔基板中培养的单个动物的定量成像方法，并将其用于阐明睡眠的分子机制。在这里，我们描述了设计、制作和成像这些载玻片所需的步骤，以及分析由此产生的行为数据。我们还描述了实验性操纵睡眠的方法。按照这些步骤，经过大约 2 小时的实验准备，我们能够以 10 或 27 µm 的空间分辨率分别同时对 24 条处于第二幼虫期到成虫期的秀丽隐杆线虫或 20 条处于第二龄期的黑腹果蝇幼虫进行成像。虽然这个系统已经被优化用于测量秀丽隐杆线虫幼虫和成虫以及黑腹果蝇幼虫的活动和静止状态，但它也可以用于在短时间或长时间内评估其他行为。此外，只需进行微小的修改，它就可以适用于广泛的小动物的行为监测。
