Background

C. elegans sleep is often assessed based on the cessation of locomotion, which is a common characteristic of sleep across the animal kingdom. Due to the intermittent nature of sleep bouts during C. elegans developmental sleep, computer vision is generally used to track the activity of C. elegans during lethargus. Animals are constrained to a single focal plane to keep them in focus. Because C. elegans can be exhausted in liquid by prolonged swimming (Ghosh and Emmons, 2008), C. elegans sleep researchers rely almost exclusively on assay formats that enforce crawling, not swimming. Also, because food may dramatically alter behavior, C. elegans sleep studies are usually undertaken in the presence of bacterial food. There are two major assay formats: animals confined to larger spaces and tracked across multiple developmental stages (Belfer et al., 2013; Nelson et al., 2013; Raizen et al., 2008) or animals confined to small spaces and tracked for shorter time intervals (Bringmann, 2011; Singh et al., 2011). The original report of C. elegans sleep utilized the first assay format, which permitted detection of sleep during each larval lethargus as animals crawled on the surface of a culture dish. This required one camera per animal, which limited throughput (Raizen et al., 2008). More recent work generally uses small chambers arranged in tight groupings, which allows simultaneous tracking of multiple animals with one camera. Various small-chamber formats are available. An automated polydimethylsiloxane (PDMS) chamber system is available with 60 chambers, but this format requires high levels of consistency in chamber loading with media and seeding bacteria, which is best achieved with robotic systems (Nelson et al., 2013). As these systems are not widely available to academic labs, most groups use other assay formats. Very small chambers constructed from agarose hydrogels have also been used to constrain locomotion of L1 larvae and it is possible that these could be reformatted for use with older C. elegans larval stages (Bringmann, 2011). But, agarose hydrogels are not easily reusable and most C. elegans sleep research focuses on the last lethargus, during the transition to adulthood. Here, we describe in detail a tractable PDMS-based, small chamber assay system, which allows simultaneous tracking of up to 10 L4 to adult animals. This is a variation of an earlier 6-chamber format assay (Singh et al., 2011). The assay is based on reusable PDMS chips and requires a minimal space to track animals in the last larval lethargus. The small chamber assay format described here requires reagents and equipment that are readily available in most C. elegans laboratories and has been adapted by several groups beyond our own.