Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2

SummaryTranscriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5–7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock

The concept of coupling in the mammalian circadian clock-network

The circadian clock network regulates daily rhythms in mammalian physiology and behavior to optimally adapt the organism to the 24-hour day/night cycle. A central pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), coordinates subordinate cellular oscillators in the brain as well as in peripheral organs to align with each other and external time. Stability and coordination of this vast network of cellular oscillators is achieved through different levels of coupling. While coupling at the molecular level and across the SCN is well-established and believed to define its function as pacemaker structure, the notion of coupling in other tissues and across the whole system is less well understood. In this review we describe the different levels of coupling in the mammalian circadian clock system - from molecules to the whole organism. We highlight recent advances in gaining knowledge of the complex organization and function of circadian network regulation and its significance for the generation of stable, but plastic intrinsic 24-hour rhythms

Working for Food Shifts Nocturnal Mouse Activity into the Day

Nocturnal rodents show diurnal food anticipatory activity when food access is restricted to a few hours in daytime. Timed food access also results in reduced food intake, but the role of food intake in circadian organization per se has not been described. By simulating natural food shortage in mice that work for food we show that reduced food intake alone shifts the activity phase from the night into the day and eventually causes nocturnal torpor (natural hypothermia). Release into continuous darkness with ad libitum food, elicits immediate reversal of activity to the previous nocturnal phase, indicating that the classical circadian pacemaker maintained its phase to the light-dark cycle. This flexibility in behavioral timing would allow mice to exploit the diurnal temporal niche while minimizing energy expenditure under poor feeding conditions in nature. This study reveals an intimate link between metabolism and mammalian circadian organization.

A novel mechanism controlling resetting speed of the circadian clock to environmental stimuli

Many aspects of mammalian physiology are driven through the coordinated action of internal circadian clocks. Clock speed (period) and phase (temporal alignment) are fundamental to an organism’s ability to synchronize with its environment. In humans, lifestyles that disturb these clocks, such as shift work, increase the incidence of diseases such as cancer and diabetes. Casein kinases 1δ and ε are closely related clock components implicated in period determination. However, CK1δ is so dominant in this regard that it remains unclear what function CK1ε normally serves. Here, we reveal that CK1ε dictates how rapidly the clock is reset by environmental stimuli. Genetic disruption of CK1ε in mice enhances phase resetting of behavioral rhythms to acute light pulses and shifts in light cycle. This impact of CK1ε targeting is recapitulated in isolated brain suprachiasmatic nucleus and peripheral (lung) clocks during NMDA- or temperature-induced phase shift in association with altered PERIOD (PER) protein dynamics. Importantly, accelerated re-entrainment of the circadian system in vivo and in vitro can be achieved in wild-type animals through pharmacological inhibition of CK1ε. These studies therefore reveal a role for CK1ε in stabilizing the circadian clock against phase shift and highlight it as a novel target for minimizing physiological disturbance in shift workers

Nocturnal and diurnal behavior assessed by the “work-for-food” protocol in small rodents

Plasticity in daily timing of activity has been observed in many species, even within an individual. The temporal phase of activity under a light–dark cycle can shift by changes in light, temperature, (perceived) predation risk, food timing, and abundance. A major determinant of the phase of locomotor activity relative to the (entrained) SCN is energy balance. In the majority of restricted feeding experiments, access to a limited amount of food is restricted to a specific time of day, thereby changing both timing of food intake and energy balance. To induce food scarcity in an ecologically appropriate way, we developed the “work-for-food” paradigm for small rodents. In this paradigm, food access is determined by wheel-running activity, and the levels of (simulated) food scarcity can therefore be titrated without imposing an externally imposed timing component. This “work-for-food” paradigm enables assessment of the effect of energy balance on the daily activity rhythms of an animal, including its decision to switch temporal niche. Adaptive behavioral strategies to cope with energetic challenges may vary depending on species, sex, age, and reproductive status. This chapter provides detailed guidelines on how to carry out the “work-for-food” paradigm as a laboratory tool to investigate (molecular) mechanisms and consequences underlying flexibility of circadian and ultradian activity patterns in small rodents. Defining the mechanisms through which metabolic feedback acts on the circadian system to shift the timing of activity relative to the light–dark cycle and entrained phase of the SCN can yield important implications for human sleep, shift-work, chronotherapy, metabolic health, and (athletic) performance.</p

Nocturnal and Diurnal Behavior Assessed by the “Work-for-Food” Protocol in Small Rodents

Plasticity in daily timing of activity has been observed in many species, even within an individual. The temporal phase of activity under a light–dark cycle can shift by changes in light, temperature, (perceived) predation risk, food timing, and abundance. A major determinant of the phase of locomotor activity relative to the (entrained) SCN is energy balance. In the majority of restricted feeding experiments, access to a limited amount of food is restricted to a specific time of day, thereby changing both timing of food intake and energy balance. To induce food scarcity in an ecologically appropriate way, we developed the “work-for-food” paradigm for small rodents. In this paradigm, food access is determined by wheel-running activity, and the levels of (simulated) food scarcity can therefore be titrated without imposing an externally imposed timing component. This “work-for-food” paradigm enables assessment of the effect of energy balance on the daily activity rhythms of an animal, including its decision to switch temporal niche. Adaptive behavioral strategies to cope with energetic challenges may vary depending on species, sex, age, and reproductive status. This chapter provides detailed guidelines on how to carry out the “work-for-food” paradigm as a laboratory tool to investigate (molecular) mechanisms and consequences underlying flexibility of circadian and ultradian activity patterns in small rodents. Defining the mechanisms through which metabolic feedback acts on the circadian system to shift the timing of activity relative to the light–dark cycle and entrained phase of the SCN can yield important implications for human sleep, shift-work, chronotherapy, metabolic health, and (athletic) performance

Cold and hunger induce diurnality in a nocturnal mammal

The mammalian circadian system synchronizes daily timing of activity and rest with the environmental light-dark cycle. Although the underlying molecular oscillatory mechanism is well studied, factors that influence phenotypic plasticity in daily activity patterns (temporal niche switching, chronotype) are presently unknown. Molecular evidence suggests that metabolism may influence the circadian molecular clock, but evidence at the level of the organism is lacking. Here we show that a metabolic challenge by cold and hunger induces diurnality in otherwise nocturnal mice. Lowering ambient temperature changes the phase of circadian light-dark entrainment in mice by increasing daytime and decreasing nighttime activity. This effect is further enhanced by simulated food shortage, which identifies metabolic balance as the underlying common factor influencing circadian organization. Clock gene expression analysis shows that the underlying neuronal mechanism is downstream from or parallel to the main circadian pacemaker (the hypothalamic suprachiasmatic nucleus) and that the behavioral phenotype is accompanied by phase adjustment of peripheral tissues. These findings indicate that nocturnal mammals can display considerable plasticity in circadian organization and may adopt a diurnal phenotype when energetically challenged. Our previously defined circadian thermoenergetics hypothesis proposes that such circadian plasticity, which naturally occurs in nocturnal mammals, reflects adaptive maintenance of energy balance. Quantification of energy expenditure shows that diurnality under natural conditions reduces thermoregulatory costs in small burrowing mammals like mice. Metabolic feedback on circadian organization thus provides functional benefits by reducing energy expenditure. Our findings may help to clarify relationships between sleep-wake patterns and metabolic phenotypes in humans

Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light [1, 2] including circadian entrainment [3], sleep induction [4], the pupillary light response (PLR) [5], and negative masking of locomotor behavior (the acute suppression of activity in response to light) [6]. How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails [7]. Significantly, both isoforms form fully functional photopigments [7]. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors

Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light [1, 2] including circadian entrainment [3], sleep induction [4], the pupillary light response (PLR) [5], and negative masking of locomotor behavior (the acute suppression of activity in response to light) [6]. How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails [7]. Significantly, both isoforms form fully functional photopigments [7]. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors