Extraordinary behavioral entrainment following circadian rhythm bifurcation in mice

Abstract

The mammalian circadian timing system uses light to synchronize endogenously generated rhythms with the environmental day. Entrainment to schedules that deviate significantly from 24 h (T24) has been viewed as unlikely because the circadian pacemaker appears capable only of small, incremental responses to brief light exposures. Challenging this view, we demonstrate that simple manipulations of light alone induce extreme plasticity in the circadian system of mice. Firstly, exposure to dim nocturnal illumination (<0.1 lux), rather than completely dark nights, permits expression of an altered circadian waveform wherein mice in light/dark/light/dark (LDLD) cycles “bifurcate” their rhythms into two rest and activity intervals per 24 h. Secondly, this bifurcated state enables mice to adopt stable activity rhythms under 15 or 30 h days (LDLD T15/T30), well beyond conventional limits of entrainment. Continuation of dim light is unnecessary for T15/30 behavioral entrainment following bifurcation. Finally, neither dim light alone nor a shortened night is sufficient for the extraordinary entrainment observed under bifurcation. Thus, we demonstrate in a non-pharmacological, non-genetic manipulation that the circadian system is far more flexible than previously thought. These findings challenge the current conception of entrainment and its underlying principles, and reveal new potential targets for circadian interventions.

Enhanced Circadian Entrainment in Mice and Its Utility under Human Shiftwork Schedules

“…Each of these animals remained very well-adapted to the light-dark cycles throughout the experiment, as evident from little activity in the light, and no visible free-running components. A measure of equality of division of activity between alternate scotophases in LDLD, the Bifurcation Symmetry Index (BSI; [17]), indicated that mice were well-bifurcated during the first phase of the experiment (0.71 ± 0.08 and 0.56 ± 0.11 for females and males respectively; t(10.0) = 1.14; p = 0.28). A complementary measure that quantifies behavioral adaptation in T-cycles, Entrainment Quotient (EQ; [17]), showed that mice were likewise well-adapted in T30 baseline (0.96 ± 0.02 and 0.83 ± 0.06 for females and males) and T36 baseline (0.97 ± 0.01 and 0.91 ± 0.05 females and males).…”

“…A measure of equality of division of activity between alternate scotophases in LDLD, the Bifurcation Symmetry Index (BSI; [17]), indicated that mice were well-bifurcated during the first phase of the experiment (0.71 ± 0.08 and 0.56 ± 0.11 for females and males respectively; t(10.0) = 1.14; p = 0.28). A complementary measure that quantifies behavioral adaptation in T-cycles, Entrainment Quotient (EQ; [17]), showed that mice were likewise well-adapted in T30 baseline (0.96 ± 0.02 and 0.83 ± 0.06 for females and males) and T36 baseline (0.97 ± 0.01 and 0.91 ± 0.05 females and males). Considering data from the two non-24 h conditions, EQ-values were significantly higher in females than males (F(1,28) = 5.93; p < 0.05), but did not differ between T30 and T36 (F(1,28) = 1.32; p = 0.26), and there was no interaction between T-cycle and sex (F(1,28) = 0.77; p = 0.39).…”

“…13 h of light, 5 h dark; LD13:5 or T18LD) and LDLD (e.g. LDLD5:4:5:4 or T18LDLD) cycles (see methods for explanation of nomenclature) [14,17,18]. Under those cycles that have been carefully characterized, behavioral adaptation reflects bona fide entrainment.…”

“…Notably, an explanation of simple masking for entrained behavior has been rejected. For example, following both LDLD and extreme T-cycles there is phase control in complete darkness (DD) [14,17,20] and reorganized rhythms of core body temperature [14,21]. In LDLD conditions alone, further evidence includes persistence of biphasic behavior in skeleton cycles [13], enhanced phase-resetting [16,22], entrainment after-effects [20], biphasic melatonin secretion [23], and altered c-Fos and clock gene expression in the Suprachiasmatic nucleus (SCN) [22,24,25] On the other hand, entrainment to extreme T-cycles lacks the characteristic large phase angle modulation [18] and period after-effects predicted by classical non-parametric entrainment theory [26,27].…”

“…Second, following rhythm bifurcation under dimly lit nights, hamsters almost immediately adjusted to light:dark (LD) shifts to any phase [16]. In hamsters and mice, the range of entrainment to LDLD cycles was greatly expanded to include 18-30 h zeitgeber periods [14,17,18]. In at least some cases, this enhanced entrainability after bifurcation persisted without the continuation of dim night-time lighting [17].…”