Expression of opioid receptors during the tail bud stage.

Opioid receptors: zfmor (A, C and E), zfdor1 (G, I and K) and zfdor2 (M, O and Q) were expressed during the tail bud stage (approximately 10 hpf). In all panels, the anterior aspect is to the left. For each opioid receptor, the left and right sides represent the embryos untreated and treated with cocaine respectively. A and B, lateral views (applies to G and H, M and N). C-F, animal pole views (applies to I-L, O-R). Arrowhead points to the future region of the tail bud, arrow indicates CNS development. Asterisks show the polster. Scale bar: 250 μm in A (applies to B-R).

Spatial distribution of zfmor at 24 and 48 hpf.

Lateral views (A-D, G-J), dorsal views (E-F, K-L). zfmor was found in the telencephalon, diencephalon, midbrain (optic tectum), MHB, hindbrain, spinal cord, eye and somites. The expression in embryos exposed to cocaine exposure was increased in the optic tectum and MHB (Asterisk and arrow, respectively). At 48 hpf, zfmor was expressed to a similar extent to what was observed at 24 hpf, and also in the MLF, notochord, pectoral flipper and heart. Cocaine induced a decrease in zfmor levels in the telencephalon and MLF (arrow and asterisk, respectively). Abbreviations: T: telencephalon; Mb: midbrain, Hb: hindbrain, D: diencephalon, N: notochord; S: somite; pf: pectoral flipper; H: heart. Scale bar: 200 μm in A (Applies to B, G and H) and 250 μm in C (Applies to D, E, F, I, J, K and L).

Spatial distribution of zfdor1 at 24 and 48 hpf.

Lateral views (A-D, G-J), dorsal views (E-F, K-L). zfdor1 was found in telencephalon, diencephalon, midbrain (optic tectum), MHB, hindbrain, spinal cord, eye and somites. Expression in embryos exposed to cocaine in the optic tectum (asterisk), telencephalon, midbrain, hindbrain and spinal. At 48 hpf zfdor1 was expressed to a similar extent as at 24 hpf, but also with expression in the MLF, notochord, pectoral flipper and heart. Cocaine induced a decrease in expression in the telencephalon and MLF (arrowhead and asterisk, respectively). Abbreviations: T: telencephalon; Mb: midbrain, Hb: hindbrain, D: diencephalon, N: notochord; SC: spinal cord; S: somite; pf: pectoral flipper; H: heart. Scale bar: 200 μm in A (Applies to B, G and H) and 250 μm in C (Applies to D, E, F, I, J, K and L).

Spatial distribution of zfdor2 at 24 and 48 hpf.

Lateral views (A-D, G-J), dorsal views (E-F, K-L). At 24 hpf, zfdor2 was found in the telencephalon, diencephalon, midbrain (optic tectum), MHB, hindbrain, spinal cord, eye and somites. At 48 hpf, zfdor2 was expressed to a similar extent as at 24 hpf, also with expression in the MLF, notochord, pectoral flipper and heart. Cocaine did not induce evident changes in the expression of zfdor2 at 24 hpf (except in the spinal cord, where its expression was increased (arrowhead), or at 48 hpf. Abbreviations: T: telencephalon; Mb: midbrain, Hb: hindbrain, D: diencephalon, N: notochord; SC: spinal cord; S: somite; pf: pectoral flipper; H: heart. Scale bar: 200 μm in A (Applies to B, G and H) and 250 μm in C (Applies to D, E, F, I, J, K and L).

Spatial distribution of dre-let-7d at 24 and 48 hpf.

Lateral views (A-D, G-J), dorsal views (E-F, K-L). dre-let-7d was found in the telencephalon, diencephalon, midbrain (optic tectum), MHB, hindbrain, spinal cord, eye and somites. Expression in embryos exposed to cocaine was observed in the telencephalon, MHB, and hindbrain (asterisk, arrowhead and arrow, respectively). At 48 hpf, dre-let-7d was expressed to a similar extent to the situation at 24 hpf, but also with expression in the MLF, notochord, pectoral flipper and heart. Cocaine induced an increase in expression in the telencephalon and MLF (arrowhead and asterisk, respectively). Abbreviations: T: telencephalon; Mb: midbrain, Hb: hindbrain, D: diencephalon, N: notochord; S: somite; pf: pectoral flipper; H: heart. Scale bar: 200 μm in A (Applies to B, G and H) and 250 μm in C (Applies to D, E, F, I, J, K and L).

Analyses of proliferation and apoptosis in whole-mount embryos by IHC and TUNEL assays.

Lateral views of embryos not exposed to cocaine are on the left side (A, C, E and G) and those exposed to the drug are on the right (B, D, F and H). Cocaine-exposed embryos showed an increase in apoptotic cells (p53) at 24 hpf (A vs. B) and a decrease at 48 hpf (C vs. D). This finding was similar with the TUNEL assay method at 24 hpf (E vs. F) and 48 hpf (G vs. H). The number of proliferating cells (in percent) (H3P) is increased both at 24 and 48 hpf (A vs. B and C vs. D, respectively). I, J and K show the percent changes in H3P, p53, and TUNEL at 24 and 48 hpf. Abbreviations: H3P: phosphorylated histone-3; p53: p53 protein; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling; T: telencephalon; Mb: midbrain; Hb: hindbrain. Scale bars 150 μm. P-Values were calculated by two-tail unpaired student′s t test: *P<0.05, **P<0.01.