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Fig. 3 Genetic and pharmacological inhibition of NPVF signaling increases locomotor activity and decreases sleep. (A–D) npvf -/- animals are more active during the day and night (A,B), and sleep less at night (C,D) compared to their npvf +/+ siblings. npvf +/- animals show an intermediate sleep phenotype that is not significantly different than npvf -/- siblings. (E–L) Pharmacological inhibition of NPVF signaling by the NPFFR1/2 antagonists RF9 and GJ-14 increased locomotor activity (E,F,I,J) and decreased sleep (G,H,K,L) during the day and night compared to DMSO control-treated siblings. Mean ± SEM from one representative experiment (A,C,E,G,L,K), three pooled experiments (F,H,J,L), or six pooled experiments (B,D) are shown. White and black bars under behavioral traces indicate day (14 h) and night (10 h), respectively. n = number of animals. *p<0.05; ***p<0.005 by One-way ANOVA with Holm-Sidak test (B,D) and Student’s t-test (F,H,J,L).