A coupled model between circadian, cell-cycle, and redox rhythms reveals their regulation of oxidative stress.

Most organisms possess three biological oscillators, circadian clock, cell cycle, and redox rhythm, which are autonomous but interact each other. However, whether their interactions and autonomy are beneficial for organisms remains unclear. Here, we modeled a coupled oscillator system where each oscillator affected the phase of the other oscillators. We found that multiple types of coupling prevent a high H2O2 level in cells at M phase. Consequently, we hypothesized a high H2O2 sensitivity at the M phase and found that moderate coupling reduced cell damage due to oxidative stress by generating appropriate phase relationships between three rhythms, whereas strong coupling resulted in an elevated cell damage by increasing the average H2O2 level and disrupted the cell cycle. Furthermore, the multicellularity model revealed that phase variations among cells confer flexibility in synchronization with environments at the expense of adaptability to the optimal environment. Thus, both autonomy and synchrony among the oscillators are important for coordinating their phase relationships to minimize oxidative stress, and couplings balance them depending on environments.

Aripiprazole disrupts cellular synchrony in the suprachiasmatic nucleus and enhances entrainment to environmental light-dark cycles in mice.

Many patients with psychiatric conditions, such as bipolar disorder and major depressive disorder, frequently experience disruptions in their sleep-wake cycles. Several case studies and clinical trials have shown that the administration of aripiprazole, a commonly prescribed antipsychotic drug, alleviates the symptoms of circadian sleep disorders in these patients. This improvement may be attributed to the effects of aripiprazole on the circadian central clock, specifically the hypothalamic suprachiasmatic nucleus (SCN), which regulates various circadian physiological rhythms, including the sleep-wake cycle, in mammals. To examine whether aripiprazole facilitates adaptation to changes in the light-dark cycle, we orally administered aripiprazole to mice and subjected them to jet-lag experiments. Mice receiving aripiprazole were more rapidly entrained to 6 h advanced light-dark cycles. Moreover, we examined the effect of aripiprazole on the cellular rhythms of SCN slice cultures and found that aripiprazole disrupted cellular synchronization in the SCN, thereby accelerating the damping of the SCN rhythm at the population level. Adenosine 3'5' monophosphate (cAMP) assay using a bioluminescence indicator revealed that intracellular cAMP level in the SCN increased following aripiprazole treatment. However, this increase was blocked by pre-treatment with the serotonin 1A receptor (5-HT1AR) antagonist. Based on these findings, we propose that aripiprazole modulates intracellular signaling, including 5-HT1AR-mediated cAMP signaling, and desynchronizes SCN neurons, ultimately leading to enhanced entrainment to phase advanced light-dark cycles in mice. These findings indicate that the improvement in sleep symptoms reported in patients with psychiatric disorders receiving aripiprazole may be due to modulation of the circadian clock. Our study provides novel insights into the potential clinical applications of aripiprazole in patients with various circadian sleep disorders.

Singularity response reveals entrainment properties in mammalian circadian clock.

Entrainment is characterized by phase response curves (PRCs), which provide a summary of responses to perturbations at each circadian phase. The synchronization of mammalian circadian clocks is accomplished through the receipt of a variety of inputs from both internal and external time cues. A comprehensive comparison of PRCs for various stimuli in each tissue is required. Herein, we demonstrate that PRCs in mammalian cells can be characterized using a recently developed estimation method based on singularity response (SR), which represents the response of desynchronized cellular clocks. We confirmed that PRCs can be reconstructed using single SR measurements and quantified response properties for various stimuli in several cell lines. SR analysis reveals that the phase and amplitude after resetting are distinguishable among stimuli. SRs in tissue slice cultures reveal tissue-specific entrainment properties. These results demonstrate that SRs can be employed to unveil entrainment mechanisms with diverse stimuli in multiscale mammalian clocks.

Analysis of circadian rhythm components in EEG/EMG data of aged mice.

Aging disrupts circadian clocks, as evidenced by a reduction in the amplitude of circadian rhythms. Because the circadian clock strongly influences sleep-wake behavior in mammals, age-related alterations in sleep-wake patterns may be attributable, at least partly, to functional changes in the circadian clock. However, the effect of aging on the circadian characteristics of sleep architecture has not been well assessed, as circadian behaviors are usually evaluated through long-term behavioral recording with wheel-running or infrared sensors. In this study, we examined age-related changes in circadian sleep-wake behavior using circadian components extracted from electroencephalography (EEG) and electromyography (EMG) data. EEG and EMG were recorded from 12 to 17-week-old and 78 to 83-week-old mice for 3 days under light/dark and constant dark conditions. We analyzed time-dependent changes in the duration of sleep. Rapid eye movement (REM) and non-REM (NREM) sleep significantly increased during the night phase in old mice, whereas no significant change was observed during the light phase. The circadian components were then extracted from the EEG data for each sleep-wake stage, revealing that the circadian rhythm in the power of delta waves during NREM sleep was attenuated and delayed in old mice. Furthermore, we used machine learning to evaluate the phase of the circadian rhythm, with EEG data serving as the input and the phase of the sleep-wake rhythm (environmental time) as the output. The results indicated that the output time for the old mice data tended to be delayed, specifically at night. These results indicate that the aging process significantly impacts the circadian rhythm in the EEG power spectrum despite the circadian rhythm in the amounts of sleep and wake attenuated but still remaining in old mice. Moreover, EEG/EMG analysis is useful not only for evaluating sleep-wake stages but also for circadian rhythms in the brain.

Optogenetic induction of hibernation-like state with modified human Opsin4 in mice.

We recently determined that the excitatory manipulation of Qrfp-expressing neurons in the preoptic area of the hypothalamus (quiescence-inducing neurons [Q neurons]) induced a hibernation-like hypothermic/hypometabolic state (QIH) in mice. To control the QIH with a higher time resolution, we develop an optogenetic method using modified human opsin4 (OPN4; also known as melanopsin), a G protein-coupled-receptor-type blue-light photoreceptor. C-terminally truncated OPN4 (OPN4dC) stably and reproducibly induces QIH for at least 24 h by illumination with low-power light (3 µW, 473 nm laser) with high temporal resolution. The high sensitivity of OPN4dC allows us to transcranially stimulate Q neurons with blue-light-emitting diodes and non-invasively induce the QIH. OPN4dC-mediated QIH recapitulates the kinetics of the physiological changes observed in natural hibernation, revealing that Q neurons concurrently contribute to thermoregulation and cardiovascular function. This optogenetic method may facilitate identification of the neural mechanisms underlying long-term dormancy states such as sleep, daily torpor, and hibernation.

Application of time lags between light and temperature cycles for growth control based on the circadian clock of Lactuca sativa L. seedlings.

The circadian clock plays an important role in agriculture, especially in highly controlled environments, such as plant factories. However, multiple environmental factors have an extremely high degree of freedom, and it is difficult to experimentally search for the optimal design conditions. A recent study demonstrated that the effect of time lags between light and temperature cycles on plant growth could be predicted by the entrainment properties of the circadian clock in Arabidopsis thaliana. Based on this prediction, it was possible to control plant growth by adjusting the time lag. However, for application in plant factories, it is necessary to verify the effectiveness of this method using commercial vegetables, such as leaf lettuce. In this study, we investigated the entrainment properties of the circadian clock and the effect of the time lag between light and temperature cycles on circadian rhythms and plant growth in Lactuca sativa L. seedlings. For evaluation of circadian rhythms, we used transgenic L. sativa L. with a luciferase reporter in the experiment and a phase oscillator model in the simulation. We found that the entrainment properties for the light and temperature stimuli and the effects of time lags on circadian rhythm and growth were similar to those of A. thaliana. Moreover, we demonstrated that changes in growth under different time lags could be predicted by simulation based on the entrainment properties of the circadian clock. These results showed the importance of designing a cultivation environment that considers the circadian clock and demonstrated a series of methods to achieve this.

Unstable Phase Response Curves Shown by Spatiotemporal Patterns in the Plant Root Circadian Clock.

Phase response curves (PRCs) play important roles in the entrainment of periodic environmental cycles. Measuring the PRC is necessary to elucidate the relationship between environmental cues and the circadian clock. Conversely, the PRCs of plant circadian clocks are unstable due to multiple factors such as biotic/abiotic noise, individual differences, changes in amplitude, growth stage, and organ/tissue specificity. However, evaluating the effect of each factor is important because PRCs are commonly obtained by determining the response of many individuals, which include different amplitude states and organs. The plant root circadian clock spontaneously generates a spatiotemporal pattern called a stripe pattern, whereby all phases of the circadian rhythm exist within an individual root. Therefore, stimulating a plant root expressing this pattern enables phase responses at all phases to be measured using an individual root. In this study, we measured PRCs for thermal stimuli using this spatiotemporal pattern method and found that the PRC changed asymmetrically with positive and negative temperature stimuli. Individual differences were observed for weak but not for strong temperature stimuli. The root PRC changed depending on the amplitude of the circadian rhythm. The PRC in the young root near the hypocotyl was more sensitive than those in older roots or near the tip. Simulation with a phase oscillator model revealed the effect of measurement and internal noises on the PRC. These results indicate that instability in the entrainment of the plant circadian clock involves multiple factors, each having different characteristics. These results may help us understand how plant circadian clocks adapt to unstable environments and how plant circadian clocks with different characteristics, such as organ, age, and amplitude, are integrated within individuals.

The singularity response reveals entrainment properties of the plant circadian clock.

Circadian clocks allow organisms to synchronize their physiological processes to diurnal variations. A phase response curve allows researchers to understand clock entrainment by revealing how signals adjust clock genes differently according to the phase in which they are applied. Comprehensively investigating these curves is difficult, however, because of the cost of measuring them experimentally. Here we demonstrate that fundamental properties of the curve are recoverable from the singularity response, which is easily measured by applying a single stimulus to a cellular network in a desynchronized state (i.e. singularity). We show that the singularity response of Arabidopsis to light/dark and temperature stimuli depends on the properties of the phase response curve for these stimuli. The measured singularity responses not only allow the curves to be precisely reconstructed but also reveal organ-specific properties of the plant circadian clock. The method is not only simple and accurate, but also general and applicable to other coupled oscillator systems as long as the oscillators can be desynchronized. This simplified method may allow the entrainment properties of the circadian clock of both plants and other species in nature.

Time Lag Between Light and Heat Diurnal Cycles Modulates CIRCADIAN CLOCK ASSOCIATION 1 Rhythm and Growth in Arabidopsis thaliana.

Plant growth responses to cues such as light, temperature, and humidity enable the entrainment of the circadian rhythms with diurnal cycles. For example, the temperature variations between day and night affect plant growth and accompany the time lag to light cycle. Despite its importance, there has been no systematic investigation into time lags, and the mechanisms behind the entrainment of the circadian rhythms with multiple cycles remain unknown. Here, we investigated systemically the effects of the time lag on the circadian rhythm and growth in Arabidopsis thaliana. To investigate the entrainment status of the circadian clock, the rhythm of the clock gene CIRCADIAN CLOCK ASSOCIATION 1 (CCA1) was measured with a luciferase reporter assay. As a result, the rhythm was significantly modulated by the time lag with +10°C heating for 4 h every day but not -10°C cooling. A model based on coupled cellular oscillators successfully described these rhythm modulations. In addition, seedling growth depended on the time lag of the heating cycle but not that of the cooling cycle. Based on the relationship between the CCA1 rhythms and growth, we established an estimation method for the effects of the time lag. Our results found that plant growth relates to the CCA1 rhythm and provides a method by which to estimate the appropriate combination of light-dark and temperature cycles.

Multicellularity enriches the entrainment of Arabidopsis circadian clock.

The phase response curve (PRC) of the circadian clock provides one of the most significant indices for anticipating entrainment of outer cycles, despite the difficulty of making precise PRC determinations in experiments. We characterized the PRC of the Arabidopsisthaliana circadian clock on the basis of its phase-locking property to variable periodic pulse perturbations. Experiments revealed that the PRC changed remarkably from continuous to discontinuous fashion, depending on the oscillation amplitude. Our hypothesis of amplitude-dependent adaptability to outer cycles was successfully clarified by elucidation of this transition of PRC as a change in the collective response of the circadian oscillator network. These findings provide an essential criterion against which to evaluate the precision of PRC measurement and an advanced understanding of the adaptability of plant circadian systems to environmental conditions.