Clock-dependent and system-driven oscillators interact in the suprachiasmatic nuclei to pace mammalian circadian rhythms

Abstract

Circadian clocks drive biological rhythms with a period of approximately 24 hours and keep in time with the outside world through daily resetting by environmental cues. While this external entrainment has been extensively investigated in the suprachiasmatic nuclei (SCN), the role of internal systemic rhythms, including daily fluctuations in core temperature or circulating hormones remains debated. Here, we show that lactating mice, which exhibit dampened systemic rhythms, possess normal molecular clockwork but impaired rhythms in both heat shock response gene expression and electrophysiological output in their SCN. This suggests that body rhythms regulate SCN activity downstream of the clock. Mathematical modeling predicts that systemic feedback upon the SCN functions as an internal oscillator that accounts for in vivo and ex vivo observations. Thus we are able to propose a new bottom-up hierarchical organization of circadian timekeeping in mammals, based on the interaction in the SCN between clock-dependent and system-driven oscillators.

Fig 1. Longitudinal telemetric monitoring of locomotor activity and body temperature in female mice.

A. Daytime (open circles) and night-time (closed circles) counts in locomotion throughout a reproductive cycle. B. Average day-night amplitude in body temperature throughout a reproductive cycle. (mean±sem, n = 3 mice; temperature amplitudes were significantly smaller every days of lactation as compared to the period before mating, F(21, 42) = 9.333, p<0.0001 2-way ANOVA Tukey's multiple comparisons test). C and D. Representative example of 24-hour variations in body temperature, recorded in a same mouse before mating (C) and during lactation (D). ZT0 defined as time of lights on.

Fig 2. Voluntary running-wheel behavior in virgin and lactating mice.

A-D. Reduced activity during lactation, under light-dark conditions. Representative actograms from virgin (A) and lactating (B) female mice. Quantification of wheel counts during daytime (C) and night-time (D). Differences were considered significant for p < 0.05, two-tailed unpaired t-test. E-H. Noticeable rhythmic organization of behavior under constant darkness. Representative actograms (E and F) and corresponding periodograms (G and H) showing a major power peak in the circadian range for both virgin (E and G) and lactating (F and H) females (the calculated free-running period was 23.72±0.01 hr and 23.77±0.14 hr, for virgin and lactating females, respectively, p>0.71 two-tailed unpaired t-test, n = 4 mice for each condition). Note that low levels in wheel counts and the presence of pups in the cage prevent the measurement of a reliable free-running period for lactating mice.

Fig 3. The circadian clockwork is preserved in the SCN of lactating mice.

A. Expression profiles of circadian clock and clock-related genes in the SCN of virgin (black) and lactating (red) mice, as assessed by quantitative PCR (mean ± SEM, n = 4 samples for each time point. The sine lines represent the best cosinor fit for each dataset (see also Table 1) B. Representative micrographs (left panels) of immunostaining for PER2 in the SCN from virgin (top) and lactating (bottom) mice, at ZT 0 and ZT 12. The number of PER2-immunopositive cells per SCN was quantified over a complete daily cycle (right panel, mean ± SEM, n = 3 mice for each time point). A highly significant time effect was observed (p < 0.0001), with no difference between reproductive states (p = 0.12, two-way ANOVA). C. Representative recordings of PER2::LUC expression showing robust circadian oscillations in SCN slices from both virgin (left panel) and lactating (right panel) females. The time of occurrence of the circadian oscillation peak during the interval between 12 and 36 hours in culture, did not differ between both conditions (25.89±0.97 hrs, n = 8, and 24.87±0.45 hrs, n = 7, respectively, p = 0.17, unpaired t-test).

Fig 4. Altered rhythmic expression of putative HSF1-target genes in the SCN of lactating mice, (mean ± SEM, n = 4, same samples and analysis as for Fig 3A, see also Table 1).

Fig 5. The daily rhythm in SCN electrical properties is suppressed in lactating mice.

A-B. Cumulative distributions of membrane potentials measured in patch-clamped cells, from virgin (A) and lactating (B) mice. C-D. Cumulative distributions of extracellular single neuron firing frequencies, from virgin (C) and lactating (D) mice. SCN slices were recorded during either daytime (empty symbols) or night-time (filled symbols). The firing frequencies measured in patch-clamped cells are shown in S1 Fig. Differences were considered significant for p < 0.05, Kolmogorov-Smirnov test, n = 30 to 48 cells, from at least 5 different mice for each condition.

Fig 6. Modeling the regulation of SCN outputs by systemic feedback.

A. Diagram depicting functional interactions entered into the mathematical model. B. The apparent free-running period of the locomotor rhythm (solid blue line) depends on the values of t and of the intrinsic period of the circadian clock. This example is constructed with numerical values published by Reinke et al. [31], with the clock period set at 23.68 h (dotted red line). For an apparent period of 23.05 h, the value of t is 22.86 h (dotted black lines). The dotted blue line represents the line y = x. C. Predicted rhythmicity of the SCN electrical output when systemic feedback is suppressed, as in SCN slices. The reduced amplitude of the SCN output recapitulates dampened rhythms observed in lactating and Hsf1-/- mice [35]. D. The suppression of clock oscillations (red line) recapitulated the rebound-shape pattern of SCN firing (green arrow) and pre-dark locomotor behavior (blue arrow) observed in Cry1-/- Cry2-/- mice [2, 36]. E. Phase-dissociation between clock oscillations and overt rhythms after abrupt phase-shift of C, as in the case of a 6-hour advance of the light schedule [37]. (Red dots and blue lines represent the middle of the up-state of C and the onset of L, respectively.)