Sleep-active neurons depolarize during sleep to suppress wakefulness circuits. Wakeactive 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 Caenorhabditis 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 interneuronswhich 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 suggest 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 forward (PVC) and reverse (including RIM) circuit activity overlap. 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 control may have evolved from locomotion control. 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.
18 19 Inhibitory sleep-active neurons depolarize at sleep onset to shut down the activity of 20 wakefulness circuits. Wake-active arousal neurons in turn suppress inhibitory sleep-active 21 neurons, thus forming a bipartite flip-flop switch. However, how sleep states are switched 22is unclear because neural circuits that directly depolarize inhibitory sleep-active neurons 23 are not understood. Using optogenetics, we solved the presynaptic circuit for depolarization 24 of the sleep-active RIS neuron in C. elegans. Surprisingly, we found that the PVC forward 25 command interneuron, which is known to control wake behavior, is a major activator of 26 RIS. The PVCs are inhibited by reverse command interneurons, which are stimulated by 27 arousing cues. This suggests a model for sleep switch operation in which declining arousal 28 increases activation of PVC, thus triggering activation of RIS. Depolarization of RIS in 29 turn promotes the activation of PVC, thus forming a positive feedback loop for all-or-none 30 sleep induction. The flip-flop sleep switch in C. elegans thus is tripartite and requires 31 excitatory sleep-promoting neurons activated by wakefulness that act as an amplifier that 32 translates reduced arousal into the depolarization of an inhibitory sleep-active neuron. A 33 tripartite flip-flop switch likely also underlies sleep state switching in other animals 34 including in mammals. 35 36 of the circuit mechanisms that determine when and how much inhibitory sleep-active 62 neurons depolarize[8, 10]. 63 64 Circuits control the depolarization of inhibitory sleep-active neurons. For example, wake-65 active wake-promoting neurons promote arousal and suppress inhibitory sleep-active 66 neurons, whereas sleep need causes tiredness and inhibitory sleep-active neuron 67 4 depolarization. Thus, inhibitory sleep-active sleep-promoting and wake-active wake-68 promoting neurons form a flip-flop switch, which ensures that sleep and wake exist as 69 discrete states. This sleep switch is under the control of arousal that favors wake and 70 inhibits sleep through the suppression of sleep-active neurons by inhibitory wake-active 71 neurons[8, 11]. It has been proposed that sleep induction is favored by disinhibition of 72 inhibitory sleep-active neurons [12-14], and also excitatory sleep-active neurons exist that 73 might perhaps present activators of inhibitory sleep-active neurons [15]. However, the 74 forces and mechanisms that flip the sleep switch from wake to sleep when an organism 75 gets tired cannot be satisfactorily explained by the present circuit models as it is unclear 76 how inhibitory sleep-active neurons are turned on when the system is set to sleep. 77 78 Sleep is under circadian and homeostatic controls that determine the timing of sleep and 79 ensure that enough of this essential physiological state takes place[16]. Sleep homeostasis 80 comprises multiple mechanisms that act on different time scales. On long time scales sleep 81 is a function of prior wakefulness, i.e. prolonged wakefulness leads to...
Within the field of sleep research, it is well established that all organisms, which possess a nervous system, need to sleep. This underlines the severity of sleep functions. In humans, sleep is essential for memory function, immune system function and energy conservation. However, none of these functions explain why sleep induces a change in consciousness.To answer these and other remaining questions about sleep, C. elegans is the optimal model organism. It offers the opportunity to study sleep in a very simple environment.Adult hermaphrodites have only 302 neurons. The connectivity of all neurons is known. Furthermore, its complete genome is sequenced. Finally, its transparency and its easy genetic tractability allow for the application of almost all known imaging methods and tools to manipulate its behavior.In my thesis, I focused on the quiescence behavior taking place throughout the development of C. elegans, which I will be referring to as sleep or lethargus.Lethargus takes places at the end of each of the four larval stages. Despite its simplicity, sleep in C. elegans displays an astonishing amount of similarities to mammalian systems. In mammals, wake-active and sleep-active brain regions mutually inhibit each other in a so-called flip-flop switch. In C. elegans, the single interneuron RIS was proven to be sleep-active. Similarly to mammalian systems, high RIS activity dampens the activity of the whole nervous system in the worm.What is not known about RIS are the neuronal networks controlling it. To shed light on that question former colleagues and I screened through all RIS presynaptic neurons using the optogenetic tools ReaChR and ArchT. Their optogenetic depolarization and hyperpolarization revealed that RIS presynaptic neurons differ in their effect on RIS. Amongst all RIS presynaptic neurons, PVC neurons were identified as activators of RIS in lethargus and RIM as modulators of RIS activity in lethargus. Both PVC and RIM neurons belong to the class of command locomotion interneurons. The regulation of RIS by command locomotion interneurons allows a direct link of sleep to locomotion, arousal and homeostasis. v A side project of my thesis, aimed for the identification of potential suppressors of the aptf-1 mutant phenotype. Aptf-1 mutants fail to immobilize in lethargus. After EMS mutagenesis, two suppressor candidates were successfully isolated according to their ability to immobilize in lethargus. The identification of candidate genes is still under research.Taken together, the presented work reveals a complex regulation of RIS in lethargus by its directly presynaptic neurons. The fact that even such a simple organism has a highly complex neuronal network for sleep regulation, strengthens the choice of C. elegans as the best model organism for sleep research. With the vast amount of available tools, not only it allows for the identification of RIS-regulating neurons, like PVC and RIM neurons, but it also opens the door for a closer understanding of regulatory pathways upstream of PVC and RIM neuron...
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