Resetting the brain clock: time course and localization of mPER1 and mPER2 protein expression in suprachiasmatic nuclei during phase shifts

Abstract

The mechanism whereby brief light pulses reset the mammalian circadian clock involves acute Per gene induction. In a previous study we investigated light-induced expression of mPer1 and mPer2 mRNA in the suprachiasmatic nuclei (SCN), with the aim of understanding the relationship between gene expression and behavioural phase shifts. In the present study, we examine the protein products of mPer1 and mPer2 genes in the core and shell region of SCN for 34 h following a phase-shifting light pulse, in order to further explore the molecular mechanism of photic entrainment. The results indicate that, during the delay zone of the phase response curve, while endogenous levels of mPER1 and mPER2 protein are falling, a light pulse produces an increase in the expression of both proteins. In contrast, during the advance zone of the phase response curve, while levels of endogenous mPER1 and mPER2 proteins are rising, a light pulse results in a further increase in mPER1 but not mPER2 protein. The regional distribution of mPER1 and mPER2 protein in the SCN follows the same pattern as their respective mRNAs, with mPER1 expression in the shell region of SCN correlated with phase advances and mPER2 in the shell region correlated with phase delays.

Fig. 1

Distribution of mPER1 and mPER2 in the SCN at CT14. Micrographs of mPER1 and mPER2 staining in the SCN in serial adjacent sections arranged from rostral to caudal. mPER1 and mPER2 showed similar regional distributions in the SCN. Scale bar, 500 μm.

Fig. 2

mPER1 and mPER2 expression after a phase-delaying light pulse. Animals were given a light pulse (LP) at CT16, then killed 2, 4, 6 or 8 h after the beginning of the light pulse. (A) Quantitative analysis of the number of mPER1-ir (left panels) and mPER2-ir (right panels) cells in LP+ and control (LP−) groups. The data are presented as mean ± SEM (*p<0.05, t-test). (B) Topographic (upper level) and quantitative (lower level) analysis of mPER1 and mPER2 in the SCN at 4 h in an LP+ and a control animal. In LP+ animals, the greatest increase in mPER1-ir cells occurs in the core SCN, while the increase of mPER2-ir cells is greatest in the shell SCN. Scale bar, 100 μm.

Fig. 3

mPER1 and mPER2 expression after a phase-advancing light pulse. Animals were given a light pulse (LP) at CT22, then killed 4, 6, 8, 10, 16, 20, 30 or 34 h after the beginning of the light pulse (data from 16 to 34 h not shown). (A) Quantitative analysis of the number of mPER1-ir (left panel) and mPER2-ir (right panel) cells in LP+ and control (LP−) groups. The data are presented as mean ± SEM (*P<0.05, t-test). (B) Topographic (upper level) and quantitative (lower level) analysis of mPER1 and mPER2 in the SCN at 10 h in an LP+ and an LP− animal. Increased numbers of mPER1-ir cells were seen in each compartment of the SCN in LP+ animals. Statistically significant changes in the number of mPER2-ir cells were not observed in either core or shell of the SCN. Scale bar, 100 μm.