(A,B) 6-dpf Tg(npvf:eGFP); Tg(npvf:GCaMP6s-P2A-tdTomato) and (C,D) Tg(npvf:ReaChR-mCitrine); Tg(npvf:GCaMP6s-P2A-tdTomato) animals were analyzed for normalized GCaMP6s fluorescence in IRa neurons before and after optogenetic stimulation of NPVF neurons. Positive (red) or negative (blue) normalized GCaMP6s fluorescence responses of individual IRa neurons is 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. The horizontal dashed line indicates division of the IRa into anterior and posterior halves. A, anterior; P, posterior; L, lateral; M, medial. ‘0’ along the A/P axis indicates location of the most posterior IRa neurons. ‘0’ along the M/L axis indicates the midline of the IRa neuron population. (E–L) Normalized GCaMP6s fluorescence of individual neurons in the anterior or posterior halves of the IRa shown as heat maps (E–H), in which each horizontal line represents an IRa neuron, and as line graphs (I–L) showing individual (gray lines) and mean (dotted line) IRa neuron responses before and after optogenetic stimulation (red lines) in Tg(npvf:eGFP); Tg(tph2:GCaMP6s-P2A-tdTomato) (E,G,I,J) and Tg(npvf:ReaChR-mCitrine); Tg(tph2:GCaMP6s-P2A-tdTomato) (F,H,K,L) animals. F1-F4 in (E–H) indicate neurons from four different fish. (M) Box plot of normalized GCaMP6s ΔF/F0 % values in anterior or posterior IRa neurons averaged for the first 10 imaging frames after stimulation of NPVF neurons in Tg(npvf:eGFP); Tg(tph2:GCaMP6s-P2A-tdTomato) (black) and Tg(npvf:ReaChR-mCitrine); Tg(npvf:GCaMP6s-P2A-tdTomato) (gray) animals. n = number of neurons quantified from four animals of each genotype. ***p<0.001, Two-way ANOVA, Holm-Sidak test.