Skip to main content
. 2017 Nov 6;6:e25727. doi: 10.7554/eLife.25727

Figure 5. Optogenetic stimulation of NPVF neurons promotes sleep.

(A) ISH of NPVF neurons (arrowhead) in a 5-dpf brain. Ventral (A) and side (A’) views are shown. a, anterior; p, posterior; d, dorsal. (B) Live image of a 5-dpf Tg(npvf:EGFP) zebrafish with brightfield and EGFP overlay. White box demarcates region shown in subsequent images. (C) EGFP and npvf coexpression shown using EGFP immunohistochemistry (IHC) and npvf FISH in a 43.4 µm thick image stack. (D) npvf and vglut2a coexpression shown using FISH in a 2.7 µm thick image stack. (E) mCitrine and npvf coexpression in a Tg(npvf:ReaChR-mCitrine) zebrafish shown using EGFP IHC and npvf FISH in a 2.7 µm thick image stack. (F–H) Tg(npvf:ReaChR-mCitrine) and WT siblings were exposed to the same blue light stimulus used in (I–L). (G,H) 75.1 ± 4.1% of NPVF neurons expressed c-fos in Tg(npvf:ReaChR-mCitrine) animals (***p<0.0001; One-way ANOVA with Dunnett’s test to sham), in contrast to similarly stimulated WT siblings (~1%, n.s., not significant) (F,H), or to sham-treated transgenic animals (~1%). (I–L) Optogenetic stimulation of NPVF neurons decreased locomotor activity and increased sleep in transgenic animals compared to WT siblings. Data are from one representative experiment (I,K), or three pooled experiments (J,L). Activity and sleep of transgenic animals are normalized to WT and represented as mean ± SEM (J,L). n = number of animals. ***p<0.0001 by Student’s t-test. Scale: 50 μm (A,B), 20 μm (C,D), 10 μm (G). See also Table 1 and Figure 5—figure supplements 1,2.

Figure 5.

Figure 5—figure supplement 1. NPVF neuronal projections.

Figure 5—figure supplement 1.

(A) A 101 μm thick maximum intensity projection image of a 5-dpf Tg(npvf:EGFP) brain. Imaging plane is denoted in blue in schematic at bottom left. Bracketed areas are magnified in (B,C). NPVF neurons have dense projections within the hypothalamus (red arrowheads) (B), sparse projections to the forebrain (A,A’) and tectum (A’), projections that converge on the raphe nuclei in the hindbrain (green arrowhead) (C), and sparse projections that extend down the spinal cord (yellow arrowhead) (A). Scale: 50 μm (A), 20 μm (B,C). Te = telencephalon; TeO = tectum; Hyp = hypothalamus; MB = midbrain; Hb = hindbrain; A = anterior; p=posterior; L = lateral; D = dorsal; V = ventral.
Figure 5—figure supplement 2. Chemogenetic stimulation of NPVF neurons promotes sleep.

Figure 5—figure supplement 2.

(A) TRPV1-TagRFP-T expression in NPVF neurons of a 5-dpf Tg(npvf:EGFP);Tg(npvf:KalTA4);Tg(UAS:TRPV1-TagRFP-T) animal shown using IHC for EGFP and TagRFP-T in a 1.1 μm thick image stack. Locomotor activity and sleep in 6–8-dpf Tg(npvf:KalTA4) and Tg(npvf:KalTA4);Tg(UAS:TRPV1-TagRFP-T) siblings treated with DMSO vehicle (B–E) or 2 μM Csn (H–K) starting at 100-hpf until the end of the experiment. Mean ± SEM for one representative experiment (B,C,H,I) or 4 (D,E) or 5 (J,K) pooled experiments are shown. White and black bars under behavioral traces indicate day (14 h) and night (10 h), respectively. (F) Following 48 hr of treatment with 2 μM Csn, 100% more c-fos expression is observed in NPVF neurons of 6-dpf Tg(npvf:EGFP);Tg(npvf:KalTA4);Tg(UAS:TRPV1-TagRFP-T) animals than their Tg(npvf:EGFP);Tg(npvf:KalTA4) siblings, shown using EGFP IHC and c-fos FISH in a representative 2.6 µm thick image stack, and quantified in (G). Yellow and white arrowheads indicate NPVF neurons with and without bright c-fos puncta, respectively. (L) npvf ISH in a 2.4 µm thick image stack of animals treated with 2 μM Csn for 48 h. There was no significant difference in the number of EGFP-positive neurons between Tg(npvf:EGFP);Tg(npvf:KalTA4) and Tg(npvf:EGFP);Tg(npvf:KalTA4);Tg(UAS:TRPV1-TagRFP-T) siblings, assayed using EGFP IHC. Data presented as mean ± SEM. n = number of animals. n.s. = not significant. **p<0.01,***p<0.005 by Student’s t-test. Scale: 20 μm (A), 5 μm (F,L).