Taken together, these data expand our understanding of the phylogenetic extent of sleep and reinforce the idea that sleep evolved early in the evolutionary history of animals. However, additional studies on other types of animals are required for a comprehensive understanding of the origin(s) and evolution of sleep.

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Study Objectives Sleep is a prominent behavioral and biochemical state observed in all animals studied, including platyhelminth flatworms. Investigations into the biochemical mechanisms associated with sleep – and wakefulness – are important for understanding how these states are regulated and how that regulation changed with the evolution of new types of animals. Unfortunately, beyond a handful of vertebrates, such studies on invertebrates are rare. Methods We investigated the effect of seven neurotransmitters, and one pharmacological compound, that modulate either sleep or wakefulness in mammals, on flatworms (Girardia tigrina). Flatworms were exposed via ingestion and diffusion to four neurotransmitters that promote wakefulness in vertebrates (acetylcholine, dopamine, glutamate, histamine), and three that induce sleep (adenosine, GABA, serotonin) along with the H1 histamine receptor antagonist pyrilamine. Compounds were administered over concentrations spanning three to five orders of magnitude. Flatworms were then transferred to fresh water and video recorded for analysis. Results Dopamine and histamine decreased the time spent inactive and increased distance travelled, consistent with their wake-promoting effect in vertebrates and fruit flies; pyrilamine increased restfulness and GABA showed a non-significant trend towards promoting restfulness in a dose-dependent manner, in agreement with their sleep-inducing effect in vertebrates, fruit flies, and Hydra. Similar to Hydra, acetylcholine, glutamate, and serotonin, but also adenosine, had no apparent effect on flatworm behavior. Conclusions These data demonstrate the potential of neurotransmitters to regulate sleep and wakefulness in flatworms and highlight the conserved action of some neurotransmitters across species.

The ability of flatworms to regenerate entire brain structures, and indeed much of their body from mere fragments of the whole animal, presents the unique opportunity to observe the development of day-night rhythms in adult animals. In many animals, young are arrhythmic, and their species-specific timing of activity develops as the animal matures. In this study, we created two flatworm cohorts, housed in isolation, that were regenerating either (1) the brain in a decapitated animal, or (2) major body structures in a bisected, tailless animal. In this way, we observed how bisection influenced the level of activity and diel rhythmicity, and how these developed as each flatworm regenerated. Here, we demonstrate that intact flatworms were predominantly active at night, with peaks in activity seen in the hours after lights-off and before lights-on. While decapitated and tailless flatworms could still move, both were less active than the original animal, and both segments retained a nocturnal lifestyle. Furthermore, decapitated flatworms, once regenerated, again showed a U-shaped pattern of nocturnal activity reminiscent of the two night-time peaks seen in the original animal. These results could be used to further investigate how regeneration may affect motor control and motor output, or to further investigate the presence of a clock in the flatworm brain.

The behaviors that characterize sleep have been observed across a broad range of different species. While much attention has been placed on vertebrates (mostly mammals and birds), the grand diversity of invertebrates has gone largely unexplored. Here, we introduce the intrigue and special value in the study of sleeping platyhelminth flatworms. Flatworms are closely related to annelids and mollusks, and yet are comparatively simple. They lack a circulatory system, respiratory system, endocrine glands, a coelom, and an anus. They retain a central and peripheral nervous system, various sensory systems, and an ability to learn. Flatworms sleep, like other animals, a state which is regulated by prior sleep/wake history and by the neurotransmitter GABA. Furthermore, they possess a remarkable ability to regenerate from a mere fragment of the original animal. The regenerative capabilities of flatworms make them a unique bilaterally symmetric animal to study a link between sleep and neurodevelopment. Lastly, the recent applications of tools for probing the flatworm genome, metabolism, and brain activity make their entrance into the field of sleep research all the more timely.