Fig 1. Presynaptic neurons control the activity of the sleep-active RIS neuron.

(A) Sample trace of RIS activity and worm locomotion behavior outside of and during lethargus. RIS has no strong calcium transients outside of lethargus but shows strong activity transients during lethargus. Upon RIS activation, worms enter sleep bouts. (S1 Data, Sheet 1A). (B) Presynaptic neurons activate or inhibit RIS outside of and during lethargus. For statistical calculations, neural activities before the stimulation period (0–0.95 min) were compared to activity levels during the stimulation period (1–1.95 min). *p < 0.05, **p < 0.01, ***p < 0.001, Wilcoxon signed rank test. (S1 Data, Sheet 1B). (C) RIS activity decreases upon optogenetic PVC and RIM hyperpolarization. Statistical calculations were performed as described in panel B, but in experiments in which SDQL was stimulated, baseline activity levels were calculated over the time interval from 0.6 to 0.95 min. Baseline activity levels were calculated starting from 0.6 min as baseline activity levels were instable before that time point. *p < 0.05, **p < 0.01, Wilcoxon signed rank test. (S1 Data, Sheet 1C). (D) Circuit model of the RIS presynaptic regulatory network. Activating synaptic input is shown as green arrows, inhibitory synaptic input is shown as red arrows, and unclear synaptic input is shown as black arrow. Gap junctions are indicated as black connections. Neurons that are presynaptic to RIS present mostly activators. PVC is essential for lethargus-specific RIS activation. RIM can inhibit RIS through tyramine and FLP-18 and can activate RIS with glutamate. ΔF/F, change of fluorescence over baseline; FLP-18, FMRF-Like Peptide 18; GCaMP, genetically encoded calcium indicator; n.s., not significant; PVC, Posterior Ventral cord neuron class name; RIM, Ring Interneuron M class name; RIS, Ring Interneuron S class name; SDQL, Posterior lateral interneuron class name—left cell.