A tripartite flip-flop sleep circuit switches sleep states

Abstract

Sleep-active neurons depolarize during sleep to suppress wakefulness circuits. Wake-active wake-promoting neurons in turn shut down sleep-active neurons, thus forming a bipartite flip-flop switch. However, how sleep is switched on is unclear because it is not known how wakefulness is translated into sleep-active neuron depolarization when the system is set to sleep. Using optogenetics in C. elegans, we solved the presynaptic circuit for depolarization of the sleep-active RIS neuron during developmentally-regulated sleep, also known as lethargus. Surprisingly, we found that RIS activation requires neurons that have known roles in wakefulness and locomotion behavior. The RIM interneurons, which are active during and can induce reverse locomotion, play a complex role and can act as inhibitors of RIS when they are strongly depolarized and as activators of RIS when they are modestly depolarized. The PVC command interneurons, which are known to promote forward locomotion during wakefulness, act as major activators of RIS. The properties of these locomotion neurons are modulated during lethargus. The RIMs become less excitable. The PVCs become resistant to inhibition and have an increased capacity to activate RIS. Separate activation of neither the PVCs nor the RIMs appears to be sufficient for sleep induction; instead, our data suggests that they act in concert to activate RIS. Forward and reverse circuit activity is normally mutually exclusive. Our data suggest that RIS may be activated at the transition between forward and reverse locomotion states, perhaps when both reverse (including RIM) and forward (PVC) circuit activity overlaps. While RIS is not strongly activated outside of lethargus, altered activity of the locomotion interneurons during lethargus favors strong RIS activation and thus sleep. The control of sleep-active neurons by locomotion circuits suggests that sleep may have evolved from locomotion quiescence. The flip-flop sleep switch in C. elegans thus requires an additional component, wake-active sleep-promoting neurons that translate wakefulness into the depolarization of a sleep-active neuron when the worm is sleepy. Wake-active sleep-promoting circuits may also be required for sleep state switching in other animals including in mammals.