Localization of oprm1 mRNA expression in the brain of zebrafish. Schematic coronal drawing of the brain of zebrafish showing the distribution of oprm1 mRNA expression. (A) Lines on a schematic sagittal drawing view of the zebrafish brain indicate levels of coronal sections, (B–N) coronal sections showing the distribution of oprm1 mRNA. n = 3, Scale bars: B–G (100 μm) and H–N (200 μm).

Expression of oprm1 mRNA in the brain of zebrafish. Photomicrographs showing expression of oprm1 genes in sagittal (A,B) and coronal sections (C–H) of zebrafish brain. (A) Antisense probe for oprm1 gene showed positive signal and (B) Sense probes for oprm1 gene showed no signals. (C) Medial zone of dorsal telencephalic area, (D) Dorsal and Ventral nucleus of ventral telencephalic area, (E,F) Anterior and posterior part of parvocellular preoptic area, (G) Habenula area, and (H) Periventricular hypothalamic area. N = 3, Scale bars: A–B (100 μm) and C–H (50 μm).

Relative oprm1 and npas4a mRNA expression in the brain. (A–G) Graphs showing the relative gene expression (fold change) of oprm1 and npas4a mRNA expression after exposure to acute vehicle (Control) and morphine via water immersion (Treated) across several brain region. (A) Schematic sagittal drawing of macro-dissected brain region, (B) olfactory bulb region, (C) Telencephalon region, (D) Hypothalamic region, (E) Cerebellum, (F) Optic tectum, (G) Spinal cord region. Data are presented as mean ± SEM. Independent t-test comparisons between control and morphine-treated fish. *P < 0.05; **P < 0.01 vs. controls (n = 6).

Effect of morphine on oprm1 mRNA expression in the brain of zebrafish. Fish were individually immersed in morphine 2 mg/L (treated) or water (control) for 20 min and then the brain samples were collected. (A–D′) Photomicrographs showing expression of oprm1 genes. (A; control and A′; morphine treated) Medial zone of the dorsal telencephalic area and dorsal nucleus of the ventral telencephalic area, (B,B′) ventral nucleus of the ventral telencephalic area, (C,C′) posterior part of preoptic area, and (D,D′) ventral periventricular hypothalamus. N = 6 each group. Scale bars: (A–D) and (A′-D′) (100 μm).

Expression of npas4a gene in the brain of zebrafish. Photomicrographs showing the mRNA expression of neural activity marker, npas4. (A) Lines on the schematic sagittal drawing view of the zebrafish brain indicate levels of coronal sections (B; control and B′; morphine treated) medial zone of dorsal telencephalic area, (C,C′) ventral nucleus of ventral telencephalic area, (D,D′) preoptic area, (E,E′) habenula, and (F,F′) ventral periventricular hypothalamus. N = 6 each group. Scale bars: (B–F) and (B′-F′) (100 μm).

Number of npas4 mRNA expressing cells in control and morphine-treated fish. (A–F) Graphs showing the difference in npas4a mRNA expression via cell counting after exposure to acute vehicle (control) and morphine via water immersion. (A) Medial zone of the dorsal telencephalic area, (B) ventral nucleus of the ventral telencephalic area, (C,D) anterior and posterior part of parvocellular preoptic area, (E) habenula, and (F) ventral periventricular hypothalamus. Data are presented as mean ± SEM. Independent t-test comparisons between control and morphine-treated fish. *P < 0.05; **P < 0.01, ***P < 0.001 vs. controls (n = 6).