Figure 1.
Per3 is a highly rhythmic circadian clock gene in whole brains in vivo and in vitro.
A) Zebrafish were kept on 2 days of 14:10 LD followed by 2 days of DD, and brains dissected at the times indicated. RNA was extracted and qPCR was performed to evaluate the relative expression of per3 mRNA. In LD there was a peak at ZT3 and trough at ZT15 (p<0.0001, One way ANOVA, n = 7–10). In DD there was also a peak at CT3 and trough at CT15 (p<0.0001, One way ANOVA, n = 3–5). The statistical significance is shown from the post-hoc Dunnett's multiple comparison test, which used the calibrator day 1 ZT15 for LD conditions, and day 4 CT15 for DD conditions. B) Whole brains were dissected, cultured and kept on a 14:10 LD cycle for 3 days. Samples were collected at the times indicated, RNA extracted and qPCR performed to determine the expression of per3 mRNA. There was a peak at ZT3, and a trough between ZT15 and ZT 21 (p<0.001, One-way ANOVA, n = 3–4). The statistical significance is shown from the Dunnett's multiple comparison post-hoc test are, using the trough, ZT21, on day 5 as the calibrator. C) Whole brains were dissected as above, cultured, but this time maintained on a 14:10 LD cycle for one day before being placed into constant darkness for two additional days. Samples were collected at the times indicated, mRNA extracted, and qPCR performed to measure the levels of per3. The rhythm persisted in per3 for one cycle under free-running conditions in vitro before damping on the second cycle in the dark. The above white black bars represent the lighting conditions, with the different plotted histogram shades representing light, dark or subjective dark phases.
Figure 2.
Per3 rhythms in all isolated regional brain cultures from per3-luc zebrafish show entrainment to LD cycles and free-running in DD.
Brains were dissected from adult per3-luc zebrafish and monitored for bioluminescence in either (A, C, E, G, J) 6 days of 12:12LD, 4 days of DD and 4 days back into LD, or (B, D, F, H, K) 7 days of 12:12LD followed by five cycles of 12:12DL. The mean bioluminescence in counts per second (CPS) is plotted (n = 3–4). All brain regions entrain to a 24-hour period in the LD cycle with a peak around ZT4–6 and free-run with a longer period in DD (p<0.0001, two-tailed paired t-test, n = 3–4 per tissue). All regions rapidly entrain to a new LD cycle or DL cycle with a peak at ZT3-5. Black and white boxes indicate the lighting regime and arrows indicate when this changes.
Figure 3.
Regional per3 expression in the adult zebrafish brain.
Adult zebrafish were kept on a 14:10LD cycle and brains were collected at ZT3 and ZT15. In situ hybridization was performed to show expression of per3 mRNA. A) Schematics of the brain containing the diencephalon, mesencephalon and rhombencephalon are shown. At ZT3 there is expression of per3 in the i) PPp and SCN, ii) PGZ, TL, Valgra and Vamgra, iii) CM, Hc and Hd, iv) EG, CCegra, and LCagra. At ZT15 there is either low or undetectable levels of per3 in these same regions. B) The antisense (AS) probe shows the per3 expression and the sense (S) control shows the background signal.
Figure 4.
Expression of light sensitive genes, cry1a and per2, in the adult zebrafish brain.
A & C) Wild type adult zebrafish were kept in the dark for 3 days and then either exposed to light for 3 hours or remained in the dark. The zebrafish were killed at CT22 and their brain and hearts dissected, RNA extracted and qPCR performed. The majority of samples collected from different brain regions showed the light responsive genes, cry1a and per2, were increased in light pulsed tissues compared to dark control. Numerous brain parts showed an increase in cry1a and per2 in the light pulsed samples (p<0.0001 and p = 0.0015 respectively, Two way ANOVA, n = 3). The light pulsed zebrafish had significantly higher cry1a and per2 levels in both the heart and brain (p<0.002, Two way ANOVA, n = 3). B & D) Wild type adult zebrafish brain parts, whole brains and hearts were cultured in L15-media for four days. Samples were exposed to a 3 hour light pulse or kept in the dark and collected at CT22, RNA extracted, and qPCR performed. Cry1a and per2 is induced by light in both the brain and heart (p<0.0001, Two way ANOVA, n = 3–5). The light pulsed brain part cultures had significantly higher cry1a and per2 levels than the dark controls (p<0.0001 and p = 0.0013 respectively, Two way ANOVA, n = 3). E) Wild type adult zebrafish were kept in the dark for 3 days and then either exposed to light for 3 hours or remained in the dark. The zebrafish were killed at CT22 and their brain dissected, fixed, frozen and sectioned. Chromogenic in situ hybridisation was performed to determine the location of per2 mRNA expression. There was minimal or undetectable expression in the dark samples. In the light pulsed samples expression of per2 was increased in the i) PPp and SCN, ii) PGZ, TL, Valgra and Vamgra, iii) Hc and Hd, iv) EG, CCegra, and LCagra.
Figure 5.
Expression of c-fos in the adult zebrafish brain.
A) Zebrafish were kept on 2 days of 14:10 LD followed by 2 days of DD, and brains dissected at the times indicated. RNA was extracted and qPCR was performed to evaluate the relative expression of c-fos mRNA. In LD there was a peak at ZT1 and ZT15 on day 1 and 2, (p<0.0001, One way ANOVA, n = 4–9). The statistical significance is shown from the post-hoc Dunnett's multiple comparison test, which used the calibrator Day 1 ZT9. In DD there was a peak at CT21 on day 3 and trough at CT9 on day 3 and day 4 (p<0.001, One way ANOVA, n = 4–9). The statistical significance is shown from the post-hoc Dunnett's multiple comparison test, which used the calibrator CT9 on day 4. The above white and black bars indicate the lighting schedule, and the shades of green reflect the light, dark and subjective dark phases. B) Adult zebrafish on a 14:10LD were given a 30 minute light pulse or kept in the dark at ZT21. Brains were dissected, RNA extracted, and qPCR performed to determine levels of c-fos mRNA as an indicator of neuronal activity. C-fos expression was five-fold higher in the brains of the light pulsed zebrafish (p<0.0001, unpaired two-tailed t-test, n = 7–8). C) c-fos is induced in specific brain regions in response to a light pulse in the night. Adult zebrafish maintained on a 14L:10D LD cycle were exposed to a 30-min light pulse at ZT21 or kept in the dark. In situ hybridisation was performed on brain sections to determine the levels of c-fos mRNA. Regions that show increased c-fos expression in response to light include i) PPp and SCN, ii) TeO, TL, Valgra and Vamgra and iii) Hc and Hd. iv) There is no change in expression in the rhombencephalon. Abbreviations: PPp (dorsal pretectum), SCN (suprachiasmatic nuclei), TeO (optic tectum), TL (torus longitudinalis), Valgra (lateral valvula cerebelli), Vamgra (medial valvula cerebelli), Hc (caudal hypothalamus) and Hd (dorsal hypothalamus).