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Fig. 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.