Figure 2—figure supplement 1.

Figure 2—figure supplement 1.

6-dpf Tg(npvf:ReaChR); Tg(npvf:GCaMP6s-P2A-tdTomato) animals and Tg(npvf:eGFP); Tg(npvf:GCaMP6s-P2A-tdTomato) controls were analyzed for GCaMP6s fluorescence normalized to tdTomato fluorescence in NPVF neurons pre- and post-optogenetic stimulation of NPVF neurons. (A) Example of a 6-dpf Tg(npvf:ReaChR); Tg(npvf:GCaMP6s-P2A-tdTomato) animal showing GCaMP6s and tdTomato fluorescence in NPVF neurons. Green and magenta signals in the eyes are due to autofluorescence. White box indicates the region of the hypothalamus that was stimulated and analyzed in subsequent panels. (B) Normalized GCaMP6s fluorescence in NPVF neurons was first recorded for 25 s (20 frames) at low-laser power. NPVF neurons were then optogenetically stimulated at higher laser power, and then normalized GCaMP6s fluorescence in NPVF neurons was immediately imaged again for 25 s (20 frames) at low-laser power. (C,C’) GCaMP6s and mCitrine fluorescence in NPVF neurons recorded one frame before (C) and after (C’) optogenetic stimulation of NPVF neurons. (D–G) Positive or negative normalized GCaMP6s fluorescence responses to optogenetic stimulation of individual NPVF neurons plotted according to their spatial location along the anterior/posterior and medial/lateral axes of the brain. The size of red or blue circles indicates the magnitude of increase or decrease of normalized GCaMP6s fluorescence, respectively. A, anterior; P, posterior; L, lateral; M, medial. ‘0’ along the A/P axis indicates location of the most posterior NPVF neurons. ‘0’ along the M/L axis indicates the midline of NPVF neuron population. (H-K) Normalized GCaMP6s fluorescence of individual NPVF neurons shown as heat maps (H,I), in which each horizontal line represents an NPVF neuron, and as line graphs (J,K) showing individual (gray lines) and mean (dotted line) normalized GCaMP6s ΔF/F₀ % values before and after optogenetic stimulation (red lines) in Tg(npvf:eGFP); Tg(tph2:GCaMP6s-P2A-tdTomato) control (H,J) and Tg(npvf:ReaChR-mCitrine); Tg(tph2:GCaMP6s-P2A-tdTomato) (I,K) animals. F1-F5 in (H,I) indicate neurons from five different fish. (L) Box plot of normalized GCaMP6s ΔF/F₀ values averaged for the first 10 imaging frames post-stimulation in Tg(npvf:GCaMP6s-P2A-tdTomato) animals that also contain either a Tg(npvf:eGFP) (black) or Tg(npvf:ReaChR-mCitrine) (gray) transgene. n = number of neurons. ***p<0.0005, Mann-Whitney test. Scale: 100 μm (A), 10 μm (C’).