Animals innately follow a near-24-h cycle of rest and activity, known as the circadian rhythm, which prepares them for daily environmental changes. The endogenous rhythm in behavioral activities is maintained with remarkable precision under constant darkness, exhibiting robust periodicity over months and minimal cycle-to-cycle variation in activity phase (Pittendrigh and Daan, 1976a; Schwartz and Zimmerman, 1990). In mammals, the suprachiasmatic nucleus (SCN) serves as the central clock, orchestrating both behavioral circadian rhythms and physiological rhythms throughout the body. The SCN is a network of circadian oscillators, with single neurons and glial cells as the cellular identities, that maintain rhythmic expressions of circadian clock molecules through the transcription-translation feedback loop (TTFL).

The oscillation within a single cell is both autonomous and persistent, yet displays a variation in period across the population (Leise et al., 2012). These oscillators couple within the network to generate a synchronized oscillation, reducing period heterogeneity and facilitating high temporal precision for the circadian clock output at the organismal level (Herzog et al., 2004). The synchronization is the essential mechanism of the SCN network that transforms diverse period, phase, and amplitude of individual oscillators into predictable and coherent outputs. However, biological systems often operate in the metastable state between complete synchronization and desynchronization (Kelso, 1995). This is sometimes due to the functional needs, such as the internal representation of seasonal time within the SCN (Pittendrigh and Daan, 1976b; Myung et al., 2015; Schmal, 2023), but it can also be due to degradation of the network through aging and disruptive timing cues such as constant light (Ohta et al., 2005; Farajnia et al., 2012).

Aging is known to cause changes in the period of circadian locomotor activity (Pittendrigh and Daan, 1974). In the widely studied laboratory mouse strain C57BL, circadian activities persist through aging, with periods typically lengthening with increasing age (Davis and Menaker, 1981; Welsh et al., 1986; Possidente et al., 1995; Valentinuzzi et al., 1997). Depending on the strain and entrainment history, periods can also shorten (Pittendrigh and Daan, 1976a). Chronic illnesses often lead to changes in circadian periodicity, which can result in sleep disturbances, as seen in diseases like Alzheimer's or Huntington's (Witting et al., 1990; Aziz et al., 2010). Disruptions in circadian gene expression have been observed in animal models of chronic kidney disease (CKD) (Hsu et al., 2012). This disruption causes instability in circadian activity when in constant darkness (Myung et al., 2019). Furthermore, disturbances in circadian rhythm have been identified in conditions such as acute respiratory failure (ARF) (Yang et al., 2020) and chronic pulmonary disease (COPD) (Giri et al., 2022). In critically ill patients, there have been reports of misalignment in internal circadian rhythms (Felten et al., 2023).

Yet, within the widely recognized Kuramoto model for synchronization, explaining these changes in period remains a challenge (Acebrón et al., 2005). Emerging evidence suggests a correlation between the period and amplitude (Myung et al., 2018; del Olmo et al., 2023). As synchronization increases, so does the circadian amplitude of a clock ensemble (Schmal et al., 2018). Oscillators achieving synchronization is fundamentally about aligning their frequencies. In systems with a finite number of oscillators, it has been numerically shown that skewness in the frequency distribution can eventually alter the mean frequency of macroscopic ensemble oscillations (Peter and Pikovsky, 2018). Given the reciprocal relationship between period and frequency, we note that a symmetric period distribution, such as Gaussian, results in a skewed frequency distribution. This skewness can influence the mean period of an oscillator ensemble, particularly when the standard deviation of the period distribution is large. This may provide additional insight into the changes of circadian period under weak synchronization observed in aging and disease conditions.

It is plausible that, within the mammalian circadian system, coupling and the resulting synchronization are integral components of its design. Most discussions on synchronization assume a Gaussian distribution of periods, where synchronization occurs at the mean period. However, the formalism of the Kuramoto model predicts that synchronization is achieved at the mean frequency. This distinction might seem subtle but could carry significant implications.

The effects of the skewed frequency distribution due to period variance that we explored are for σ = 4 h. Data on the electrical firing rate and the bioluminescent clock gene reporter activity from dissociated single SCN neurons show a standard deviation of <2 h (Herzog et al., 2004; Honma et al., 2004, 2012). This amounts to <10% difference between the mean and median frequencies. This difference diminishes as the synchronization tightens the distribution (Figure 1D). When the system is coupled below the critical level, the effects of a skewed frequency distribution become apparent in externally observable states of period and amplitude. Specifically, the period systematically deviates from the mean period. Our analytical and numerical approach provides insights into these deviations.

The effect described above is closely related to another issue in chronobiology, namely the skewed Arnold tongue when presented in terms of period instead of frequency (Schmal et al., 2015, 2020). The simplest form of synchronization, commonly referred to as entrainment, is the unidirectional synchronization of an internal clock to an external Zeitgeber signal, such as rhythmic light or temperature cues. Entrainment typically occurs within a wedge-shaped entrainment region within the Zeitgeber period (T) and Zeitgeber strength (K) parameter plane that broadens for large Zeitgeber strength and tapers toward the intrinsic period (τ) for decreasing strength K. Since the information of the Zeitgeber signal typically enters the underlying system's equations via the frequency, the reciprocal relationship between frequency and period leads to an asymmetric entrainment region in the period domain. This has implications for circadian physiology as it could directly translate into asymmetric distributions of chronotypes.

In a broader biological context, our findings have potential implications for both development and disease. It is believed that the coupling within the circadian clock network changes throughout development (Olejniczak et al., 2023). Around the time of birth, the circadian period displays wide variations across species (Rivkees, 2003; Yamazaki et al., 2005; Bellavia et al., 2006), a phenomenon potentially explained by the lack of coupling (Weinert and Weiß, 1997). As aging progresses, the clock network is thought to deteriorate (Farajnia et al., 2014). Thus, the network coupling, established during embryonic and perinatal stages, might peak and then gradually decline throughout mature and senescent stages (Figure 4A, upper, “Internal states”). Although this deterioration is gradual, the switch-like relationship between coupling strength (K) and synchronization (R) implies that R will remain stable as long as K stays above a critical level. As senescence begins and coupling weakens, there may be stage-specific alterations in circadian amplitude and periods (Figure 4A, lower, “External states”). In humans, these senescence-related changes are evident among Alzheimer's disease (AD) patients, with low circadian activity amplitude and delayed acrophase (Satlin et al., 1995).

Similarly, a sudden illness will not have immediate effects on the circadian rhythm since the underlying oscillators have already been well synchronized, and they require time to gradually fall into desynchronization (Myung et al., 2012). Even if an illness were to completely disrupt the coupling, the RMS amplitude would decay gradually and the mean period would remain initially unaffected (Figure 4B). However, over time the macroscopic period will experience significant fluctuations, a phenomenon consistent with the unstable circadian period observed in chronic illnesses, such as CKD (Myung et al., 2019). A significant alteration in circadian period is also observed in AD (Volicer et al., 2012).

There is a question concerning the interpretation of the macroscopic period (τ_macro) compared to the mean internal period (τ₀). This question ties into a longstanding debate within SCN physiology regarding the preferred extracellular signal pathway: diffusive signaling (also known as volume transmission) vs. synaptic signaling (also known as wiring transmission) (Moore, 2013). The macroscopic period is estimated from the average oscillation of the entire ensemble. In the bioluminescent reporter system, this value corresponds to the whole-field luminometry data from a culture dish. The signal through volume transmission would carry information of the macroscopic period as it is the average of the entire ensemble output. In contrast, the mean internal period is calculated as the average period of each individual cellular oscillator, as determined from imaging data. From a distant tissue receiving the circadian signal through volume transmission, the detailed individual activities are unknown. Synaptic signaling originates from individual neurons, and therefore, individual periods can be accessed. In this context, the macroscopic period corresponds to the period observed in volume transmission, while the mean internal period can be evaluated through synaptic signaling. The circadian amplitude of volume transmission reflects the RMS amplitude of the clock assembly I, whereas the amplitude of wiring transmission reflects the order parameter R. These two are comparable, as seen in Figure 3. However, the variation of period at low coupling strength is much larger in the volume transmission (as reflected by the macroscopic period) than in the synaptic transmission (reflected by the mean internal period). At least for the circadian locomotor outputs, the macroscopic period from our simulation seems to better represent realistic observations, where the diffusible clock signals originate in the SCN (LeSauter and Silver, 1998) and propagate through cerebrospinal fluid irrigation (Leak and Moore, 2012).

In this study, we demonstrated that synchronization can influence the observed period of an ensemble. One potential application is assessing the synchronization state of the cell population by examining the macroscopic period of total reporter activities from cultured cells or tissues. The degree of synchronization can vary due to factors such as development, the level of integration within a tissue, or pathological conditions. Therefore, synchronization can serve as a qualitative indicator of these states, which can be gauged by the macroscopic period and/or period distribution. In principle, it is possible to evaluate the phenotype of an in vitro culture reflecting these states (Kumpošt et al., 2021). Although obtaining an exact measure of synchronization might be challenging, as indicated in Figure 4A, the macroscopic period can provide insights into developmental maturation, cellular interactions, or even the pathological phenotype of cellular ensembles modeling a disease. This becomes particularly relevant when individual periods are not directly accessible. Since synchronization can influence the macroscopic period, such determinations can be made solely by observing this period. This approach can be especially valuable in studies, for example, of spheroids.

Although the coupling strength may not be directly measurable in a given individual, our study suggests that other observable features could indicate decreased coupling. For instance, a decrease in amplitude of activities and core temperature can signal a loss of synchrony and/or coupling. It is also conceivable that the changes in the circadian period caused by decreased coupling could lead to a desynchronization of behavioral organization within the same individual, such as timings of eating and motor behaviors. These potential indicators could be used to evaluate the loss of internal synchrony or coupling, which could in turn inform the development of personalized chronotherapies, an approach that has yet to demonstrate significant benefits (Lee et al., 2021).

It is interesting to note that in our schematic simulation over the course of life, the critical coupling level to enter or exit the stable period would be crossed twice. However, the components of the network can change through aging (Farajnia et al., 2014), and it is unclear whether the critical coupling strength at these two points corresponds to the same value. It would also be important to note that the environmental factors can affect differently toward synchronization at these two points of development. These factors can be systemic, given that other peripheral clocks can influence the pacing of the master clock (Myung et al., 2018, 2019; Chrobok et al., 2022). Therefore, it would be valuable to investigate what other factors, in conjunction with the local network coupling, determine the fate of synchrony.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

JM conceptualized the study and wrote the first draft of the manuscript. JM, SH, and CS performed mathematical analysis. JM, HV, and M-YW interpreted the results. All authors discussed the results, contributed to the article and final manuscript, and approved the submitted version.