Figure 3

Effects of exposure to embryonic ethanol (0.5% v/v, 22–24 hpf) and injection of cxcl12a mRNA (50 ng/µL) on the density of branch points and terminal points in different brain areas of the projections from Hcrt neurons in the aAH, POA and pAH subpopulations of 6 dpf Hcrt:EGFP zebrafish. (A) Bar graphs (n = 4–5/group) with purple-colored bars show projection data of aAH Hcrt neurons and their branch points (left) and terminal points (right) in the SP and DP. Bar graphs (n = 4–5/group) with red-colored bars show projection data of ectopic POA Hcrt neurons and their branch points (left) and terminal points (right) in the SP and DP. Bar graphs (n = 4–5/group) with green-colored bars show projection data of pAH Hcrt neurons and their branch points (left, F (2, 10) = 17.14, p = 0.0006) and terminal points (right, F (2, 10) = 15.91, p = 0.008) in the LC. (B) Images show digital representations of Hcrt neuronal projections, created from photomicrographs (25×, dorsal view) of the Control + Vehicle-injected (left), Ethanol + Vehicle-injected (middle) and Control + cxcl12a mRNA-injected (right) zebrafish, obtained using confocal microscopy with the “Filaments” function of Imaris software. As with the bar graph data, the primarily ascending aAH Hcrt projections are shown in purple, the ascending POA Hcrt projections are shown in red and the primarily descending pAH Hcrt projections are shown in green. (C) Digitally constructed enlargements of the projections in the SP, DP, LC and LFB from the aAH (purple), POA (red) and pAH (green) Hcrt neurons are shown below, with branch points indicated by white dots and terminal points indicated by blue dots. Scale bars: low magnification 50 µm; SP: 15 µm; DP: 15 µm; LC: 10 µm; LFB: 10 µm. All results are shown as means ± standard errors. ** p < 0.01, *** p < 0.001. Abbreviations: aAH: anterior part of the anterior hypothalamus, POA: preoptic area, pAH: posterior part of the anterior hypothalamus, LFB: lateral forebrain bundle, SP: subpallium, DP: dorsal pallium, LC: locus coeruleus, Hcrt: hypocretin, hpf: hours post fertilization, dpf: days post fertilization.