Phase angle difference alters coupling relations of functionally distinct circadian oscillators revealed by rhythm splitting.

The interactions (i.e., coupling) between multiple oscillators of a circadian system determine basic properties of the integrated pacemaker. Unfortunately, there are few experimental models to investigate the putative interactions of functionally defined oscillators comprising the mammalian circadian pacemaker. Here the authors induce in hamsters a novel circadian entrainment pattern that is characterized by the daily expression of robust wheel-running activity in each scotophase of a 24-h light:dark:light:dark cycle. The daily activity bouts are mediated by 2 circadian oscillators, here designated "daytime" and "nighttime," that have been temporally dissociated under this light regime. To assess the phase dependence of interactions between oscillatory components, the phase relationship of the 2 daily scotophases was manipulated over a 4-h range, and the timing of activity of the daytime and nighttime components under entrained and probe conditions was examined. The average phase angle of entrainment and the day-to-day variability of activity onset of each activity component depended on the phase relationship of the respective scotophases and not on whether the component occurred in the daytime or the nighttime. Short-term denial of wheel access subsequently influenced amount and duration of wheel running but not timing of its onset, suggesting that only the former measures depend on a homeostatic mechanism sensitive to the time elapsed since prior intense running. Replacement of individual photophases with darkness revealed phase attraction between oscillators that was not dependent on the phase relationship of component oscillators but differed for daytime versus nighttime activity components. Entrainment patterns shown here cannot be accounted for by only nonparametric actions of light. Instead, the phase-dependent interactions of oscillators strongly influence entrainment properties, whereas intrinsic functional differences in dissociated oscillators apparently influence their attraction in darkness. This model system may be ideal for identifying genomic and physiological factors that mediate these interactions and thus contribute importantly to system properties of the mammalian circadian clock.