Two coupled circadian oscillations regulate Bmal1-ELuc and Per2-SLR2 expression in the mouse suprachiasmatic nucleus.

Circadian rhythms in clock genes, Bmal1 and Per2 expression were monitored simultaneously in the cultured slice of mouse suprachiasmatic nucleus (SCN) by dual bioluminescent reporters. In the neonatal SCN, the phase-relation between the Bmal1 and Per2 rhythms were significantly changed during culture. Medium exchange produced phase-dependent phase shifts (PRCm) in the Bmal1 rhythms, but not in the Per2 rhythms. As a result, the two circadian rhythms were temporally dissociated after medium exchange. In the adult SCN, the phase-relation between the two rhythms was kept constant during culture at least up to 20 cycles. The amplitude of PRCm in the adult SCN was significantly attenuated in the Bmal1 rhythm, whereas a PRCm was developed in the Per2 rhythm. The circadian period was not systematically affected by medium exchange in either of rhythms, regardless of whether it was in the neonatal or the adult SCN. Tetrodotoxin, a sodium channel blocker, enhanced the phase-response in both rhythms but abolished the phase-dependency. In addition, tetrodotoxin lengthened the circadian period independent of the phase of administration. Thus, the Bmal1 and Per2 rhythms in the SCN are dissociable and likely regulated by distinct circadian oscillators. Bmal1 is the component of a Bmal1/REV-ERBa/ROR loop and Per2 a Per/Cry/BMAL1/CLOCK loop. Both loops could be molecular mechanisms of the two circadian oscillators that are coupled through the protein product of Bmal1. The coupling strength between the two oscillations depends on developmental stages.