Figure 6

Effects of exposure to embryonic ethanol (0.5% v/v, 22?24 hpf) and administration of Cxcr4 antagonist AMD3100 (1 µM, 2?24 hpf) on the location of Hcrt neurons, density of cxcl12a and cxcr4b transcripts, and colocalization of cxcl12a and cxcr4b transcripts with Hcrt neurons in 28 hpf zebrafish brain. (a) Representative photomicrographs (×25) illustrate the zebrafish brain in a dorsal/ventral view with the pAH, aAH, ORR and Tel of the brain outlined by dashed lines and show that ethanol compared to control increases the number of Hcrt neurons (green) at 28 hpf only in the aAH and ectopically in the ORR, and these effects are completely blocked by AMD3100 as shown with cells counterstained with DAPI (blue). (b) Bar graphs (n = 4?5) show increased number of Hcrt neurons in the aAH of the ethanol group relative to the control and ethanol + AMD3100 groups, with no effect of ethanol on Hcrt number in the pAH. Ethanol-induced Hcrt neurons in the ORR are evident only in the ethanol group (c) Representative photomicrographs show the density of internalized cxcl12a (red, top) and cxcr4b (white, bottom) transcripts to be increased throughout the brain in the ethanol group compared to the control and ethanol + AMD3100 groups. (d) Bar graphs (n = 4?5) show increased density of the cxcl12a (top) and cxcr4b (bottom) transcripts in all brain regions, including the pAH, aAH, ORR and Tel, in the ethanol group compared to the control and ethanol + AMD3100 groups. (e) Enlargement of boxes at 28 hpf show the Hcrt neurons, located in the aAH and ectopically in the ORR but not the pAH, co-localize with a greater number of both cxcl12a (top) and cxcr4b (bottom) transcripts in the ethanol group compared to the control and ethanol + AMD3100 groups. (f) Bar graphs (n = 4?5) show an increased number of cxcl12a (top) and cxcr4b (bottom) transcripts in Hcrt neurons in the aAH of the ethanol group compared to the control and ethanol + AMD3100 groups, with no change between groups occurring in the pAH. Ectopically located Hcrt neurons in the ORR seen only in the ethanol group show similar levels of co-localization as evident in Hcrt neurons of the aAH. Scale bar low magnification 20 µm; high magnification 8 µm. *p < 0.05 compared to ethanol. Results are shown as means ± standard errors. AH anterior hypothalamus, aAH anterior part of the anterior hypothalamus, pAH posterior part of the anterior hypothalamus, ORR optic recess region, Tel Telencephalon, Hcrt Hypocretin, hpf hours post fertilization.