Entrainment of circadian clocks in mammals by arousal and food

Circadian rhythms in mammals are regulated by a system of endogenous circadian oscillators (clock cells) in the brain and in most peripheral organs and tissues. One group of clock cells in the hypothalamic SCN (suprachiasmatic nuclei) functions as a pacemaker for co-ordinating the timing of oscillators elsewhere in the brain and body. This master clock can be reset and entrained by daily LD (light-dark) cycles and thereby also serves to interface internal with external time, ensuring an appropriate alignment of behavioural and physiological rhythms with the solar day. Two features of the mammalian circadian system provide flexibility in circadian programming to exploit temporal regularities of social stimuli or food availability. One feature is the sensitivity of the SCN pacemaker to behavioural arousal stimulated during the usual sleep period, which can reset its phase and modulate its response to LD stimuli. Neural pathways from the brainstem and thalamus mediate these effects by releasing neurochemicals that inhibit retinal inputs to the SCN clock or that alter clock-gene expression in SCN clock cells. A second feature is the sensitivity of circadian oscillators outside of the SCN to stimuli associated with food intake, which enables animals to uncouple rhythms of behaviour and physiology from LD cycles and align these with predictable daily mealtimes. The location of oscillators necessary for food-entrained behavioural rhythms is not yet certain. Persistence of these rhythms in mice with clock-gene mutations that disable the SCN pacemaker suggests diversity in the molecular basis of light- and food-entrainable clocks.     

The suprachiasmatic nucleus: a clock of multiple components

Although impressive progress has been made in understanding the molecular basis of pacemaker function in the suprachiasmatic nucleus (SCN), fundamental questions about cellular and regional heterogeneity within the SCN, and how this heterogeneity might contribute to SCN pacemaker function at a tissue level, have remained unresolved. To reexamine cellular and regional heterogeneity within the SCN, the authors have focused on two key questions: which SCN cells are endogenously rhythmic and/or directly light responsive? Observations of endogenous rhythms of electrical activity, gene/protein expression, and protein phosphorylation suggest that the SCN in mammals examined to date is composed of anatomically distinct rhythmic and nonrhythmic components. Endogenously rhythmic neurons are primarily found in rostral, dorsomedial, and ventromedial portions of the nucleus; at mid and caudal levels, the distribution of endogenously rhythmic cells in the SCN has the appearance of a "shell." The majority of nonrhythmic cells, by contrast, are located in a central "core" region of the SCN, which is complementary to the shell. The location of light-responsive cells, defined by direct retinohypothalamic input and light-induced gene expression, largely overlaps the location of nonrhythmic cells in the SCN core, although, in hamsters and mice light-responsive cells are also present in the ventral portion of the rhythmic shell. While the relative positions of rhythmic and light-responsive components of the SCN are similar between species, the precise boundaries of these components, and neurochemical phenotype of cells within them, are variable. Intercellular communication between these components may be a key feature responsible for the unique pacemaker properties of the SCN observed at a tissue and whole animal level.     

Neural correlates of individual differences in circadian behaviour

Daily rhythms in mammals are controlled by the circadian system, which is a collection of biological clocks regulated by a central pacemaker within the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Changes in SCN function have pronounced consequences for behaviour and physiology; however, few studies have examined whether individual differences in circadian behaviour reflect changes in SCN function. Here, PERIOD2::LUCIFERASE mice were exposed to a behavioural assay to characterize individual differences in baseline entrainment, rate of re-entrainment and free-running rhythms. SCN slices were then collected for ex vivo bioluminescence imaging to gain insight into how the properties of the SCN clock influence individual differences in behavioural rhythms. First, individual differences in the timing of locomotor activity rhythms were positively correlated with the timing of SCN rhythms. Second, slower adjustment during simulated jetlag was associated with a larger degree of phase heterogeneity among SCN neurons. Collectively, these findings highlight the role of the SCN network in determining individual differences in circadian behaviour. Furthermore, these results reveal novel ways that the network organization of the SCN influences plasticity at the behavioural level, and lend insight into potential interventions designed to modulate the rate of resynchronization during transmeridian travel and shift work.     

Comprehensive Mapping of Regional Expression of the Clock Protein PERIOD2 in Rat Forebrain across the 24-h Day

In mammals, a light-entrainable clock located in the suprachiasmatic nucleus (SCN) regulates circadian rhythms by synchronizing oscillators throughout the brain and body. Notably, the nature of the relation between the SCN clock and subordinate oscillators in the rest of the brain is not well defined. We performed a high temporal resolution analysis of the expression of the circadian clock protein PERIOD2 (PER2) in the rat forebrain to characterize the distribution, amplitude and phase of PER2 rhythms across different regions. Eighty-four LEW/Crl male rats were entrained to a 12-h: 12-h light/dark cycle, and subsequently perfused every 30 min across the 24-h day for a total of 48 time-points. PER2 expression was assessed with immunohistochemistry and analyzed using automated cell counts. We report the presence of PER2 expression in 20 forebrain areas important for a wide range of motivated and appetitive behaviors including the SCN, bed nucleus, and several regions of the amygdala, hippocampus, striatum, and cortex. Eighteen areas displayed significant PER2 rhythms, which peaked at different times of day. Our data demonstrate a previously uncharacterized regional distribution of rhythms of a clock protein expression in the brain that provides a sound basis for future studies of circadian clock function in animal models of disease.     

Effects of vasoactive intestinal peptide genotype on circadian gene expression in the suprachiasmatic nucleus and peripheral organs

The neuropeptide vasoactive intestinal polypeptide (VIP) has emerged as a key candidate molecule mediating the synchronization of rhythms in clock gene expression within the suprachiasmatic nucleus (SCN). In addition, neurons expressing VIP are anatomically well positioned to mediate communication between the SCN and peripheral oscillators. In this study, we examined the temporal expression profile of 3 key circadian genes: Per1, Per2 , and Bmal1 in the SCN, the adrenal glands and the liver of mice deficient for the Vip gene (VIP KO), and their wild-type counterparts. We performed these measurements in mice held in a light/dark cycle as well as in constant darkness and found that rhythms in gene expression were greatly attenuated in the VIP-deficient SCN. In the periphery, the impact of the loss of VIP varied with the tissue and gene measured. In the adrenals, rhythms in Per1 were lost in VIP-deficient mice, while in the liver, the most dramatic impact was on the phase of the diurnal expression rhythms. Finally, we examined the effects of the loss of VIP on ex vivo explants of the same central and peripheral oscillators using the PER2::LUC reporter system. The VIP-deficient mice exhibited low amplitude rhythms in the SCN as well as altered phase relationships between the SCN and the peripheral oscillators. Together, these data suggest that VIP is critical for robust rhythms in clock gene expression in the SCN and some peripheral organs and that the absence of this peptide alters both the amplitude of circadian rhythms as well as the phase relationships between the rhythms in the SCN and periphery.     

Peripheral circadian oscillators in mammals: time and food

Peripheral cells from mammalian tissues, while perfectly capable of circadian rhythm generation, are not light sensitive and thus have to be entrained by nonphotic cues. Feeding time is the dominant zeitgeber for peripheral mammalian clocks: Daytime feeding of nocturnal laboratory rodents completely inverts the phase of circadian gene expression in many tissues, including liver, heart, kidney, and pancreas, but it has no effect on the SCN pacemaker. It is thus plausible that in intact animals, the SCN synchronizes peripheral docks primarily through temporal feeding patterns that are imposed through behavioral rest-activity cycles. In addition, body temperature rhythms, which are themselves dependent on both feeding patterns and rest-activity cycles, can sustain circadian, clock gene activity in vivo and in vitro. The SCN may also influence the phase of rhythmic gene expression in peripheral tissues through direct chemical pathways. In fact, many chemical signals induce circadian gene expression in tissue culture cells. Some of these have been shown to elicit phase shifts when injected into intact animals and are thus candidates for physiologically relevant timing cues. While the response of the SCN to light is strictly gated to respond only during the night, peripheral oscillators can be chemically phase shifted throughout the day. For example, injection of dexamethasone, a glucocorticoid receptor agonist, resets the phase of circadian liver gene expression during the entire 24-h day. Given the bewildering array of agents capable of influencing peripheral clocks, the identification of physiologically relevant agents used by the SCN to synchronize peripheral clocks will clearly be an arduous undertaking. Nevertheless, we feel that experimental systems by which this enticing problem can be tackled are now at hand.     

Conditioning in the Orcadian System

Light is the dominant environmental cue for entrainment of circadian rhythms. In mammals, light entrains rhythms by resetting the phase of a circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Until recently, the mechanism responsible for pacemaker resetting by light was thought to be exclusively sensitive to photic cues. New experiments indicate, however, that this mechanism is more plastic than once thought; is amenable to conditioned stimulus control; and is capable of acquiring, through conditioning, new response capabilities. These experiments showed that, in rats, a neutral stimulus paired with light in Pavlovian conditioning trials is capable of eliciting cellular and behavioral effects characteristic of circadian clock phase resetting by light, expression of Fos protein in the ventrolateral region of the SCN, and phase shifts of free-running rhythms. These novel results open up a previously unappreciated perspective on photic phase resetting and entrainment of circadian rhythms. Specifically, they suggest that the process normally initiated by light to reset the clock can be modified by learning and events in the environment that reliably precede the onset of light can assume the resetting function of light.     

Forced desynchronization of dual circadian oscillators within the rat suprachiasmatic nucleus

The circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) contains multiple autonomous single-cell circadian oscillators and their basic intracellular oscillatory mechanism is beginning to be identified. Less well understood is how individual SCN cells create an integrated tissue pacemaker that produces a coherent read-out to the rest of the organism. Intercellular coupling mechanisms must coordinate individual cellular periods to generate the averaged, genotype-specific circadian period of whole animals. To noninvasively dissociate this circadian oscillatory network in vivo, we (T.C. and A.D.-N.) have developed an experimental paradigm that exposes animals to exotic light-dark (LD) cycles with periods close to the limits of circadian entrainment. If individual oscillators with different periods are loosely coupled within the network, perhaps some of them would be synchronized to the external cycle while others remain unentrained. In fact, rats exposed to an artificially short 22 hr LD cycle express two stable circadian motor activity rhythms with different period lengths in individual animals. Our analysis of SCN gene expression under such conditions suggests that these two motor activity rhythms reflect the separate activities of two oscillators in the anatomically defined ventrolateral and dorsomedial SCN subdivisions. Our "forced desychronization" protocol has allowed the first stable separation of these two regional oscillators in vivo, correlating their activities to distinct behavioral outputs, and providing a powerful approach for understanding SCN tissue organization and signaling mechanisms in behaving animals.     

The mammalian circadian clock shop.

In mammals, a master circadian pacemaker driving daily rhythms in behavior and physiology resides in the suprachiasmatic nucleus (SCN). The SCN contains multiple circadian oscillators that synchronize to environmental cycles and to each other in vivo. Rhythm production, an intracellular event, depends on more than eight identified genes. The period of the rhythms within the SCN also depends upon intercellular communication. Many other tissues also retain the ability to generate near 24 -h periodicities although their place in the organization of circadian timing is still unclear. This paper focuses on the tissue-, cellular- and molecular-level events that generate and entrain circadian rhythms in behavior in mammals and emphasizes the apparent differences between the SCN and peripheral oscillators.     

Simulation of day-length encoding in the SCN: from single-cell to tissue-level organization

The circadian pacemaker of the SCN is a heterogeneous structure containing many single-cell oscillators that display phase differences in gene expression and electrical activity rhythms. Thus far, it is unknown how single neurons contribute to the population signal measured from the SCN. The authors used single-unit electrical activity rhythms that have previously been recorded in SCN slices and investigated in simulation studies how changes in pattern shape and distribution of single neurons alter the ensemble activity rhythm of the SCN. The results were compared with recorded ensemble rhythms. The simulations show that single units should be distributed in phase to render the recorded multiunit waveform and that different distributions can account for the multiunit pattern of the SCN, including a bimodal distribution. Vice versa, the authors show that the single-unit distribution cannot be inferred from the ensemble pattern. Photoperiodic encoding by the SCN relies on changes in waveform of the neuronal output from the SCN and received special attention in this study's simulations. The authors show that a broadening or narrowing of the multiunit pattern can be based on changes in phase differences between neurons, as well as on changes in the circadian pattern of individual neurons. However, these mechanisms give rise to differences in the maximal discharge level of the multiunit pattern, leading to testable predictions to distinguish between the 2 mechanisms. If single units broaden their activity pattern in long days, the maximum frequency of the multiunit activity should increase, while an increase in phase difference between the single-unit activity rhythms should lead to a decrement in maximum frequency. The simulations also show that coding for day-length by an evening and morning oscillator is not self-evident and will only work under a limited set of conditions in which the distribution within each component and temporal distance between the components is taken into account. While the simulations were based on single-cell and multiunit electrical activity patterns, they are also relevant for understanding the relation between single-cell and population molecular expression profiles.     

Neuropeptide signaling differentially affects phase maintenance and rhythm generation in SCN and extra-SCN circadian oscillators

Circadian rhythms in physiology and behavior are coordinated by the brain's dominant circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Vasoactive intestinal polypeptide (VIP) and its receptor, VPAC(2), play important roles in the functioning of the SCN pacemaker. Mice lacking VPAC(2) receptors (Vipr2(-/-)) express disrupted behavioral and metabolic rhythms and show altered SCN neuronal activity and clock gene expression. Within the brain, the SCN is not the only site containing endogenous circadian oscillators, nor is it the only site of VPAC(2) receptor expression; both VPAC(2) receptors and rhythmic clock gene/protein expression have been noted in the arcuate (Arc) and dorsomedial (DMH) nuclei of the mediobasal hypothalamus, and in the pituitary gland. The functional role of VPAC(2) receptors in rhythm generation and maintenance in these tissues is, however, unknown. We used wild type (WT) and Vipr2(-/-) mice expressing a luciferase reporter (PER2::LUC) to investigate whether circadian rhythms in the clock gene protein PER2 in these extra-SCN tissues were compromised by the absence of the VPAC(2) receptor. Vipr2(-/-) SCN cultures expressed significantly lower amplitude PER2::LUC oscillations than WT SCN. Surprisingly, in Vipr2(-/-) Arc/ME/PT complex (Arc, median eminence and pars tuberalis), DMH and pituitary, the period, amplitude and rate of damping of rhythms were not significantly different to WT. Intriguingly, while we found WT SCN and Arc/ME/PT tissues to maintain a consistent circadian phase when cultured, the phase of corresponding Vipr2(-/-) cultures was reset by cull/culture procedure. These data demonstrate that while the main rhythm parameters of extra-SCN circadian oscillations are maintained in Vipr2(-/-) mice, the ability of these oscillators to resist phase shifts is compromised. These deficiencies may contribute towards the aberrant behavior and metabolism associated with Vipr2(-/-) animals. Further, our data indicate a link between circadian rhythm strength and the ability of tissues to resist circadian phase resetting.     

Glucocorticoid hormones inhibit food-induced phase-shifting of peripheral circadian oscillators.

The circadian timing system in mammals is composed of a master pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus and slave clocks in most peripheral cell types. The phase of peripheral clocks can be completely uncoupled from the SCN pacemaker by restricted feeding. Thus, feeding time, while not affecting the phase of the SCN pacemaker, is a dominant Zeitgeber for peripheral circadian oscillators. Here we show that the phase resetting in peripheral clocks of nocturnal mice is slow when feeding time is changed from night to day and rapid when switched back from day to night. Unexpectedly, the inertia in daytime feeding-induced phase resetting of circadian gene expression in liver and kidney is not an intrinsic property of peripheral oscillators, but is caused by glucocorticoid signaling. Thus, glucocorticoid hormones inhibit the uncoupling of peripheral and central circadian oscillators by altered feeding time.     

Circadian organization is governed by extra-SCN pacemakers

In mammals, a pacemaker in the suprachiasmatic nucleus (SCN) is thought to be required for behavioral, physiological, and molecular circadian rhythms. However, there is considerable evidence that temporal food restriction (restricted feedisng [RF]) and chronic methamphetamine (MA) can drive circadian rhythms of locomotor activity, body temperature, and endocrine function in the absence of SCN. This indicates the existence of extra-SCN pacemakers: the Food Entrainable Oscillator (FEO) and Methamphetamine Sensitive Circadian Oscillator (MASCO). Here, we show that these extra-SCN pacemakers control the phases of peripheral oscillators in intact as well as in SCN-ablated PER2::LUC mice. MA administration shifted the phases of SCN, cornea, pineal, pituitary, kidney, and salivary glands in intact animals. When the SCN was ablated, disrupted phase relationships among peripheral oscillators were reinstated by MA treatment. When intact animals were subjected to restricted feeding, the phases of cornea, pineal, kidney, salivary gland, lung, and liver were shifted. In SCN-lesioned restricted-fed mice, phases of all of the tissues shifted such that they aligned with the time of the meal. Taken together, these data show that FEO and MASCO are strong circadian pacemakers able to regulate the phases of peripheral oscillators.     

Hierarchically coupled ultradian oscillators generating robust circadian rhythms

Ensembles of mutually coupled ultradian cellular oscillators have been proposed by a number of authors to explain the generation of circadian rhythms in mammals. Most mathematical models using many coupled oscillators predict that the output period should vary as the square root of the number of participating units, thus being inconsistent with the well-established experimental result that ablation of substantial parts of the suprachiasmatic nuclei (SCN), the main circadian pacemaker in mammals, does not eliminate the overt circadian functions, which show no changes in the phases or periods of the rhythms. From these observations, we have developed a theoretical model that exhibits the robustness of the circadian clock to changes in the number of cells in the SCN, and that is readily adaptable to include the successful features of other known models of circadian regulation, such as the phase response curves and light resetting of the phase.     

Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus (SCN) of the brain and subsidiary oscillators in most peripheral cell types. While oscillators in SCN neurons are known to function in a self-sustained fashion, peripheral oscillators have been thought to damp rapidly when disconnected from the control exerted by the SCN. Using two reporter systems, we monitored circadian gene expression in NIH3T3 mouse fibroblasts in real time and in individual cells. In conjunction with mathematical modeling and cell co-culture experiments, these data demonstrated that in vitro cultured fibroblasts harbor self-sustained and cell-autonomous circadian clocks similar to those operative in SCN neurons. Circadian gene expression in fibroblasts continues during cell division, and our experiments unveiled unexpected interactions between the circadian clock and the cell division clock. Specifically, the circadian oscillator gates cytokinesis to defined time windows, and mitosis elicits phase shifts in circadian cycles.     

Cognitive Performance as a Zeitgeber: Cognitive Oscillators and Cholinergic Modulation of the SCN Entrain Circadian Rhythms

The suprachiasmatic nucleus (SCN) is the primary circadian pacemaker in mammals that can synchronize or entrain to environmental cues. Although light exerts powerful influences on SCN output, other non-photic stimuli can modulate the SCN as well. We recently demonstrated that daily performance of a cognitive task requiring sustained periods of attentional effort that relies upon basal forebrain (BF) cholinergic activity dramatically alters circadian rhythms in rats. In particular, normally nocturnal rats adopt a robust diurnal activity pattern that persists for several days in the absence of cognitive training. Although anatomical and pharmacological data from non-performing animals support a relationship between cholinergic signaling and circadian rhythms, little is known about how endogenous cholinergic signaling influences SCN function in behaving animals. Here we report that BF cholinergic projections to the SCN provide the principal signal allowing for the expression of cognitive entrainment in light-phase trained animals. We also reveal that oscillator(s) outside of the SCN drive cognitive entrainment as daily timed cognitive training robustly entrains SCN-lesioned arrhythmic animals. Ablation of the SCN, however, resulted in significant impairments in task acquisition, indicating that SCN-mediated timekeeping benefits new learning and cognitive performance. Taken together, we conclude that cognition entrains non-photic oscillators, and cholinergic signaling to the SCN serves as a temporal timestamp attenuating SCN photic-driven rhythms, thereby permitting cognitive demands to modulate behavior.