Worms sleep: a perspective

Abstract

I review the history of sleep research in Caenorhabditis elegans, briefly introduce the four articles in this issue focused on worm sleep, and propose future directions our field might take.

During sleep we are vulnerable and unproductive. Yet, we spend one third of our lives asleep. When scientists search for sleep in other animals, including in those distantly related to us, they find it. The neuroscientist Allan Rechtschaffen famously said, “If sleep doesn’t serve an absolutely vital function, it is the greatest mistake evolution ever made” (Rechtschaffen, 1971).

I became interested in worm sleep when, in the 90s doing thesis work in Leon Avery’s lab, I noticed that RC301 males stopped moving and feeding after mating. This behavior was noted by others (LeBoeuf et al., 2014). When I returned to lab after completing clinical training, I initially dabbled in fruit fly sleep research, but ultimately returned to worms due to their faster life cycle and simpler, more transparent nervous system.

Worms stop moving and eating during lethargus, a 2–3 hour period at the transition between larval stages (Cassada and Russell, 1975; Singh and Sulston, 1978). Insightfully, Sir John Sulston noted that movement quiescence was broken up by brief bouts of motion (Singh and Sulston, 1978), suggesting that lethargus was a behavioral state. My imagination was captured by a 1999 Rougvie lab paper, showing that expression of lin-42, the C. elegans ortholog of the circadian clock gene period, oscillates during development (Jeon et al., 1999). There was a fixed phase relationship between expression of lin-42 and worm quiescence, just as there was between expression of period and circadian sleep in other animals.

Several worm biologists I chatted with (Meera Sundaram, Anne Hart, Victor Ambros, Piali Sengupta, Young-jai You, and others) had similar ideas and encouraged me to pursue the idea that worms sleep during lethargus. I presented our preliminary behavioral results on worm sleep at the 2005 LA Worm Meeting. They were well-received but with a healthy dose of skepticism. I am grateful for both the encouragement and the critiques. At that same worm meeting, I was asked to chair a parallel slide session on worm behavior. It was my first time chairing a session at a scientific meeting. I think I was more nervous than any of the speakers and had trouble focusing on the talks. But in a moment of scientific serendipity, one of the speakers was Cheryl van Buskirk, then a post-doc in the Sternberg lab. Cheryl showed a video of a worm after over-expression of the gene lin-3. There was absolutely no movement in the video! I panicked thinking this was a malfunction of AV equipment, which I had no idea how to fix. Only on returning to Philadelphia and discussing meeting highlights with Meera Sundaram and Gautam Kao did it occur to me that Cheryl’s video was in fact working but that the worm was completely quiescent. The field of “worm sleep” was born.

In the early days, we and other worm sleep groups were asked by editors and reviewers to use the term “sleep-like” instead of “sleep”. We pushed back because “sleep-like” implies it is not really sleep but a state similar to sleep. I also did not like the term because sleep itself can be very different across species. Is the behavior like sleep in brown bats, who are quiescent 18 hours each day? Is it like sleep in dolphins, who alternate sleep in the two sides of their brains? Or is it like sleep in fruit flies, whose sleep is strongly circadian?

The sociology of science can be funny. Sleep in flies was first described in 2000 (Hendricks et al., 2000; Shaw et al., 2000), eight years before worm sleep was proposed. Yet, the term “sleep” did not fully stick in the fly literature until worm sleep was described. The collective reaction of the sleep field, which was made up of mostly mammalian sleep researchers, was, “…ok ok, we can now accept that a fruit fly sleeps…but worms? no way!”. Similarly, the term “worm sleep” seemed to stick only after three Cal. Tech labs collaborated to show that jellyfish sleep (Nath et al., 2017). Maybe it will require a description of sleep in sponges in order to fully accept that jellyfish sleep? The first appearance of sleep on this planet keeps getting pushed back.

There remains some healthy scientific skepticism, but “worm sleep” is pretty well accepted now. It has also become an important model for sleep in other organisms. This is powerfully illustrated by a paper describing the first murine forward genetic screen for sleep mutants. In that screen, Funato and colleagues identified the salt-induced kinase SIK3 as a sleep regulator (Funato et al., 2016). Remarkably, to make the point of evolutionary conservation of this gene function, they showed that the worm ortholog of SIK3, called KIN-29, regulated worm sleep. (As an aside, a role for kin-29 in quiescence regulation was actually reported almost a decade earlier, by Young-jai You (van der Linden et al., 2008). She just did not call it “sleep” back then.)

The focus of the “worm sleep” field in its first 5–10 years was on developmentally timed sleep (DTS) during lethargus. But beginning with the van Buskirk’s lab’s identification of sleep in adult animals recovering from exposures such as high heat that cause cellular stress (Hill et al., 2014), the breadth of “worm sleep” has been expanding. We often refer to this latter type of sleep, which is relevant to sleepiness and fatigue during human illness, as “stress- or sickness-induced sleep” (SIS). Sleep behavior has also been described during prolonged fasts in both larvae and adults (Skora et al., 2018; Wu et al., 2018), and quiescence has been described in the setting of metabolic satiation (You et al., 2008).

This worm sleep field is still small—for every worm sleep lab, there are probably >50 fly sleep labs. Yet our field has already made tremendous progress, which, at some levels, is at least on par with progress in understanding fly and mouse sleep. This is particularly true for studies of sleep circuitry, where I would argue our understanding in worms exceeds that in other systems. These studies have converged on two second-order interneurons called RIS and ALA, identified by the Bringmann (Turek et al., 2013) and van Buskirk (Hill et al., 2014) labs, respectively. Nichols and colleagues showed that among nearly all neurons, only RIS is strongly activated during lethargus quiescent bouts (Nichols et al., 2017). While there are some differences between the roles of RIS and ALA (Konietzka et al., 2020; Robinson et al., 2019), it is likely that these are the main two neurons executing the sleep state. Both of these neurons are peptidergic: ALA secretes a cocktail of neuropeptides that include FLP-13, FLP-24, and NLP-8 to promote quiescence (Nath et al., 2016; Nelson et al., 2014) whereas RIS secretes FLP-11 (and perhaps also NLP-8) for its quiescent output (Turek et al., 2016). A current challenge in the field is to understand the mechanism of RIS and ALA activation as well as the downstream mechanisms. Several other neurons, as well as non-neural cells, have been implicated in sleep regulation (Choi et al., 2013; Choi et al., 2015; Grubbs et al., 2019; Singh et al., 2011; Skora et al., 2018) in some fashion or another but details regarding how they connect to RIS and/or ALA are still being worked out.

Each of the four worm sleep papers published in this edition of the Journal of Neurogenetics carries an important message. The van Buskirk lab paper (Goetting, et al, J. Neurogenet., in press) contributes to our understanding of the mechanism of SIS. They also describe a new trigger for SIS: skin injury, which is relevant to the human complaint of severe fatigue after an operation. The Bringmann lab paper (Busack et al, J. Neurogenet., in press) describes a method for long-term optogenetic manipulation of worms. Developing such methods is important because prior worm tools have been optimized for much shorter durations of manipulation and observations. The Nelson lab describes the role of orcokinins, neuropeptides conserved among molting animals, in regulating sleep (Honer et al, J. Neurogenet., in press). This study again emphasizes the important, but complex roles of neuropeptides in behavioral state modulation. Finally, the Hart lab paper reminds us that not all that stops moving is sleep and that we must remain self-skeptical as a field. They suggest that cessation of swimming is better explained by neuromuscular fatigue than by sleep (Schuch et al, J. Neurogenet., in press).

What does the future hold for worm sleep? Thus far, the field has primarily studied genes and neurons with large effect sizes. This has taken us far and has led to the identification of key neurons and genes. But sleep regulation is complex and there are likely numerous other genes and neurons with smaller, quantitative roles in sleep regulation. Understanding the sleep/wake circuit is a solvable problem in C. elegans using currently available optogenetic tools for manipulating and recording physiological activity. A lovely example of such a circuit-interrogation was recently published (Maluck et al., 2020). Finding new genes will require higher throughput forward genetic screens and increased reliance on quantitative measurements of quiescence.

Given the conservation of sleep, we should be able to use the worm to model human sleep disorders. A step in that direction has been reported (Huang et al., 2017). Finally, by comparing what happens during sleep and sleep curtailment in worms to other animals, we can gain insight into the ancient function of sleep and maybe understand why we cannot live without it (Anafi et al., 2019; Bennett et al., 2018; Driver et al., 2013; Fry et al., 2016; Hill et al., 2014; Wu et al., 2018).