Figure 1.
Bright-field and fluorescent images of developing embryos at different developmental stages that show Tg(Gnat2:gal4-VP16/UAS:nfsB-mCherry) transgene expression in the pineal gland and retina.
(A) At 24 hpf, mCherry was detected in the pineal gland (arrow). (B) Strong mCherry expression was seen in the pineal gland (arrows). (C–F) Between 56 and 120 hpf, mCherry was detected in both the pineal gland (downward arrows) and retina (upward arrows). The intensity of transgene expression increased during embryonic development.
Figure 2.
Cryostat sections of pineal gland and retina of transgenic zebrafish (2-month old) that show mCherry (left panels), opsin antibody immunoreactivity (middle panels), and co-localization of mCherry and opsin antibodies (right panels).
(A) mCherry and double-cone opsin immunoreactivity in the pineal gland and retina. mCherry and double-cone opsin antibodies were co-localized in the pineal and retinal photoreceptor cells. (B, C) mCherry, blue- and UV-cone opsin immunoreactivity in the pineal gland and retina. Blue-cone and UV-cone antibody immunoreactivity were not seen in the pineal gland, but were detected in the retina where they co-localized with mCherry. (D) mCherry and rhodopsin immunoreactivity in the pineal gland and retina. Rhodopsin antibody immunoreactivity was detected in both pineal and retinal rod photoreceptor cells, but did not co-localize with mCherry. (E) Fluorescent images of pineal and retinal sections that were processed without primary antibodies (negative controls).
Figure 3.
Cryostat sections of pineal gland and retina of transgenic zebrafish (1, 3 and 8 months) that show mCherry (left panels), double-cone opsin antibody (zpr-1) immunoreactivity (middle panels), and co-localization of mCherry and double-cone opsin antibody (right panels).
(A) At 1 month of age, strong transgene expression was seen in the pineal gland and retina. mCherry and zpr-1 immunoreactivity were co-localized (B) At 3 months, the expression of mCherry decreased in the retina, but increased in the pineal gland. Co-localized of mCherry and zpr-1 immunoreactivity was detected in the pineal gland and central retina. (C) By 8 months, mCherry was detected in the pineal gland, but not in the retina. (D) Fluorescent images of pineal and retinal sections (from 8-months-old animals) that were processed without primary antibodies (negative controls). Arrows point the optic nerve.
Figure 4.
Cryostat sections of pineal gland and retina of transgenic zebrafish (8-month old) that show mCherry (left panels), double-cone opsin antibody immunoreactivity (zpr-1, middle panels), and co-localization of mCherry and opsin antibody (right panels) before and after the treatment with metronidazole.
(A) Pineal and retinal sections from untreated transgenic fish. Strong mCherry expression was detected in the pineal gland, but not in the retina. zpr-1 immunoreactivity was seen in both pineal and retinal photoreceptor cells. (B) After 1 metronidazole treatment, mCherry expression and zpr-1 immunoreactivity in the pineal gland were decreased. In the retina, strong zpr-1 immunoreactivity was detected. (C) After 4 metronidazole treatments, the expression of mCherry and zpr-1 immunoreactivity in the pineal gland was completely diminished. Strong zpr-1 immunoreactivity was detected in the retina. (D) Fluorescent images of pineal and retinal sections that were processed without primary antibodies (negative controls). Dashed lines outline the retina. Arrows point to the optic nerve.
Figure 5.
TUNEL labeling of pineal and retinal sections of metronidazole-treated and untreated transgenic (Tg) and wild-type (AB) zebrafish (8 months old).
(A, B) Pineal and retinal sections of untreated transgenic fish. No obvious cell death was detected. (C, D) Pineal and retinal sections of transgenic zebrafish after 4 metronidazole treatments. In the pineal gland, TUNEL-positive cells were detected (arrows). No cell death was seen in the retina. (E–H) Pineal and retinal sections from untreated and metronidazole-treated wild-type zebrafish. No cell death was seen in the pineal gland or retina. Dashed lines outline the retina.
Figure 6.
Behavioral visual thresholds of wild-type (white bars), metronidazole-treated wild-type (gray bars), transgenic (hatched bars) and metronidazole-treated transgenic fish (black bars) measured under white, red and green illumination, respectively.
(A) Absolute visual thresholds measured at 6pm in white, red and green light in animals kept in the normal LD. Under either light conditions, no statistic differences in thresholds were seen between wild-type, metronidazole-treated wild-type, transgenic and metronidazole-treated transgenic animals. (B) Threshold difference between 3am and 7pm measured in wild-type, metronidazole-treated wild-type, transgenic and metronidazole-treated transgenic fish, respectively. The difference in visual threshold between 3am and 7pm decreased in metronidazole-treated transgenic fish tested under either light conditions. Data represent the Means ± SE (n = 12). * p<0.05; n.s., no statistic difference.
Figure 7.
Circadian rhythms of behavioral visual sensitivity in transgenic fish (circles, n = 12) and metronidazole-treated transgenic zebrafish after 25-min of light exposures in the early morning (squares; n = 12).
Threshold measurements were performed with white (A), red (B) and green light (C), respectively. Horizontal hatched and black bars on the top of the panel represent subjective day and night, respectively. Data represents the Means ± SE.
Figure 8.
Behavioral visual thresholds measured in white, red and green light, respectively, in transgenic zebrafish after metronidazole treatments.
Each circle represents a visual threshold measurement from one fish (n = 12). When measured with white light, visual thresholds increased in most times of the day and night, except at 3 and 7am, at which times the thresholds were already at the highest levels (A). Under red and green light illumination conditions, visual threshold decreased in the day and night, except 7pm, at which time the thresholds were already at the lowest levels (B, C). Dashed lines represent visual thresholds obtained from untreated control fish. Horizontal hatched and black bars on the top of the panel represent subjective day and night, respectively.
Figure 9.
Locomotor activities of control (untreated transgenic fish; white circles, n = 12) and metronidazole-treated transgenic fish (black circles, n = 12) at different times of the day and night in DD.
In the control group, the fish were more active in the day than in the night. In metronidazole-treated fish, swimming activities were similar in the day and night. Horizontal hatched and black bars on the top of the panel represent subjective day and night, respectively. Data represents the Means ± SE.