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

Abstract

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.

Weak Coupling Between Intracellular Feedback Loops Explains Dissociation of Clock Gene Dynamics

“…A similar dissociation has been observed after preparation of SCN slices, i.e., after transferring the clock system from in vivo to in vitro conditions [19,20]. Furthermore, subsequent culture medium exchanges elicit differential phase-dependent phase shifts of Per2 and Bmal1 oscillations [21]. Ubiquity of the circadian rhythms in broad biological processes and organisms suggests that, despite the common underlying mechanism of negative feedback loop, there can be diverse biological implementations [43].…”

“…Furthermore, phase response dynamics to timing of medium exchange were found to be different in Bmal1-ELuc and Per2-SLR2 oscillations in cultured slices of the SCN [21]. While Bmal1 showed a significant response to the medium exchange in neonatal mice [22], Per2 did not [21].…”

“…Furthermore, phase response dynamics to timing of medium exchange were found to be different in Bmal1-ELuc and Per2-SLR2 oscillations in cultured slices of the SCN [21]. While Bmal1 showed a significant response to the medium exchange in neonatal mice [22], Per2 did not [21].Despite these experimental observations, existing mathematical models have not taken into account such long-transient dissociation of clock genes, presumably involved in different feedback loops. We use data-driven conceptual and contextual modeling approaches to identify intracellular network topologies and parameter realizations that enable experimentally observed dissociation dynamics.…”

“…This leads again to significantly shorter Bmal1-ELuc periods across a three week long-term recording [19]. Dissociation of the two genes was observed in different reporter constructs that express luciferases with more distinct emission wavelengths [21]. Furthermore, phase response dynamics to timing of medium exchange were found to be different in Bmal1-ELuc and Per2-SLR2 oscillations in cultured slices of the SCN [21].…”

“…Dissociation of the two genes was observed in different reporter constructs that express luciferases with more distinct emission wavelengths [21]. Furthermore, phase response dynamics to timing of medium exchange were found to be different in Bmal1-ELuc and Per2-SLR2 oscillations in cultured slices of the SCN [21]. While Bmal1 showed a significant response to the medium exchange in neonatal mice [22], Per2 did not [21].Despite these experimental observations, existing mathematical models have not taken into account such long-transient dissociation of clock genes, presumably involved in different feedback loops.…”