Figure 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). See also Figure 3—figure supplements 1 and 2.

Figure 3—figure supplement 1. Genetic and pharmacological inhibition of NPVF signaling alters sleep/wake architecture.

(A) Sequence alignment of WT and mutant zebrafish NPVF preproproteins. Colored boxes demarcate predicted mature peptide sequences. The mutation results in a reading frame change after amino acid 108 (gray shading) and a premature stop codon after amino acid 146, resulting in a mutant protein lacks the RFRP2 and RFRP3 peptide sequences. (B–M) Sleep/wake architecture of npvf -/- animals (B–E) and WT animals treated with RF9 (F–I) or GJ-14 (J–M). npvf -/- animals displayed altered sleep/wake architecture compared to their npvf +/+ siblings. Similar, but more pronounced, effects were observed in RF9 or GJ-14 treated larvae compared to their DMSO treated siblings. Bar graphs show mean ± SEM from six pooled experiments (B–E) or four pooled experiments (F–M), with measurements of sleep latency (time to first sleep bout after light transitions) (B,F,J), sleep bouts per hour (C,G,K), sleep bout length (D,H,L), and wake activity (E,I,M). n = number of animals. n.s. = not significant. **p<0.01, ***p<0.005 by One-way ANOVA with Holm-Sidak test (B–E) or Student’s t-test (F–M).

Figure 3—figure supplement 2. Pharmacological inhibition of NPVF signaling suppresses RFRP-overexpression induced sleep.

(A,B,E,F) Following DMSO vehicle treatment, RFRP1,3 overexpression increased sleep in transgenic animals compared to WT siblings and to pre-HS (red bracket comparison). Following treatment with either RF9 (C,D) or GJ-14 (G,H), RFRP1,3 overexpression had no sleep effect post-HS compared to pre-HS (red bracket comparison). Yellow bars indicate heat shock. Pre- and Post-HS data is calculated for the day of HS. White and black bars under behavioral traces indicate day (14 h) and night, respectively. Mean ± SEM from one representative experiment (A,C,E,F,G), or two (H) or three (B,D) pooled experiments are shown. n = number of animals. DMSO vehicle and drug treated siblings were from the same clutch (A–D, E–H), but were tested in different videotrackers due to the numerous comparisons being made. n.s. = not significant. **p<0.01, ***p<0.005 by Two-way ANOVA with Holm-Sidak test.