An intact pituitary vasopressin system is critical for building a robust circadian clock in the suprachiasmatic nucleus.

The circadian clock is a biological timekeeping system that oscillates with a circa-24-h period, reset by environmental timing cues, especially light, to the 24-h day-night cycle. In mammals, a "central" clock in the hypothalamic suprachiasmatic nucleus (SCN) synchronizes "peripheral" clocks throughout the body to regulate behavior, metabolism, and physiology. A key feature of the clock's oscillation is resistance to abrupt perturbations, but the mechanisms underlying such robustness are not well understood. Here, we probe clock robustness to unexpected photic perturbation by measuring the speed of reentrainment of the murine locomotor rhythm after an abrupt advance of the light-dark cycle. Using an intersectional genetic approach, we implicate a critical role for arginine vasopressin pathways, both central within the SCN and peripheral from the anterior pituitary.

On the origin and evolution of the dual oscillator model underlying the photoperiodic clockwork in the suprachiasmatic nucleus.

Decades have now passed since Colin Pittendrigh first proposed a model of a circadian clock composed of two coupled oscillators, individually responsive to the rising and setting sun, as a flexible solution to the challenge of behavioral and physiological adaptation to the changing seasons. The elegance and predictive power of this postulation has stimulated laboratories around the world in searches to identify and localize such hypothesized evening and morning oscillators, or sets of oscillators, in insects, rodents, and humans, with experimental designs and approaches keeping pace over the years with technological advances in biology and neuroscience. Here, we recount the conceptual origin and highlight the subsequent evolution of this dual oscillator model for the circadian clock in the mammalian suprachiasmatic nucleus; and how, despite our increasingly sophisticated view of this multicellular pacemaker, Pittendrigh's binary conception has remained influential in our clock models and metaphors.

Inferring dynamic topology for decoding spatiotemporal structures in complex heterogeneous networks.

Extracting complex interactions (i.e., dynamic topologies) has been an essential, but difficult, step toward understanding large, complex, and diverse systems including biological, financial, and electrical networks. However, reliable and efficient methods for the recovery or estimation of network topology remain a challenge due to the tremendous scale of emerging systems (e.g., brain and social networks) and the inherent nonlinearity within and between individual units. We develop a unified, data-driven approach to efficiently infer connections of networks (ICON). We apply ICON to determine topology of networks of oscillators with different periodicities, degree nodes, coupling functions, and time scales, arising in silico, and in electrochemistry, neuronal networks, and groups of mice. This method enables the formulation of these large-scale, nonlinear estimation problems as a linear inverse problem that can be solved using parallel computing. Working with data from networks, ICON is robust and versatile enough to reliably reveal full and partial resonance among fast chemical oscillators, coherent circadian rhythms among hundreds of cells, and functional connectivity mediating social synchronization of circadian rhythmicity among mice over weeks.

Dynamics of periodically forced finite N-oscillators, with implications for the social synchronization of animal rest-activity rhythms.

The possible mechanisms for the synchronization of rest-activity rhythms of individual animals living in groups is a relatively understudied question; synchronized rhythms could occur by entrainment of individuals to a common external force and/or by social synchronization between individuals. To gain insight into this question, we explored the synchronization dynamics of populations of globally coupled Kuramoto oscillators and analyzed the effects of a finite oscillator number (N) and the variable strengths of their periodic forcing (F) and mutual coupling (K). We found that increasing N promotes entrainment to a decreasing value of F, but that F could not be reduced below a certain level determined by the number of oscillators and the distribution width of their intrinsic frequencies. Our analysis prompts some simple predictions of ecologically optimal animal group sizes under differing natural conditions.

Social synchronization of circadian rhythmicity in female mice depends on the number of cohabiting animals.

Communal animals often engage in group activities that require temporal synchrony among its members, including synchrony on the circadian timescale. The principles and conditions that foster such collective synchronization are not understood, but existing literature hints that the number of interacting individuals may be a critical factor. We tested this by recording individual circadian body temperature rhythms of female house mice housed singly, in twos (pairs), or in groups of five (quintets) in constant darkness; determining the daily phases of the circadian peak for each animal; and then calculating the cycle-to-cycle phase relationship between cohabiting animals over time. Significant temporal coherence was observed in quintets: the proportion of quintets (4/7), but not pairs (2/8), that became synchronized was greater than could be achieved by the complete simulated reassortment of all individuals. We speculate that the social coupling of individual circadian clocks of group members may be adaptive under certain conditions, and we propose that optimal group sizes in nature may depend not only on species-specific energetics, spatial behaviour and natural history but also on the mathematics of synchronizing assemblies of weakly coupled animal oscillators.

Social forces can impact the circadian clocks of cohabiting hamsters.

A number of field and laboratory studies have shown that the social environment influences daily rhythms in numerous species. However, underlying mechanisms, including the circadian system's role, are not known. Obstacles to this research have been the inability to track and objectively analyse rhythms of individual animals housed together. Here, we employed temperature dataloggers to track individual body temperature rhythms of pairs of cohabiting male Syrian hamsters (Mesocricetus auratus) in constant darkness and applied a continuous wavelet transform to determine the phase of rhythm onset before, during, and after cohabitation. Cohabitation altered the predicted trajectory of rhythm onsets in 34% of individuals, representing 58% of pairs, compared to 12% of hamsters single-housed as 'virtual pair' controls. Deviation from the predicted trajectory was by a change in circadian period (τ), which tended to be asymmetric-affecting one individual of the pair in nine of 11 affected pairs-with hints that dominance might play a role. These data implicate a change in the speed of the circadian clock as one mechanism whereby social factors can alter daily rhythms. Miniature dataloggers coupled with wavelet analyses should provide powerful tools for future studies investigating the principles and mechanisms mediating social influences on daily timing.

Distinct patterns of Period gene expression in the suprachiasmatic nucleus underlie circadian clock photoentrainment by advances or delays.

The circadian clock in the mammalian hypothalamic suprachiasmatic nucleus (SCN) is entrained by the ambient light/dark cycle, which differentially acts to cause the clock to advance or delay. Light-induced changes in the rhythmic expression of SCN clock genes are believed to be a critical step in this process, but how the two entrainment modalities--advances vs. delays--engage the molecular clockwork remains incompletely understood. We investigated molecular substrates of photic entrainment of the clock in the SCN by stably entraining hamsters to T cycles (non-24-h light/dark cycles) consisting of a single 1-h light pulse repeated as either a short (23.33-h) or a long (24.67-h) cycle; under these conditions, the light pulse of the short cycle acts as "dawn," whereas that of the long cycle acts as "dusk." Analyses of the expression of the photoinducible and rhythmic clock genes Period 1 and 2 (Per1 and Per2) in the SCN revealed fundamental differences under these two entrainment modes. Light at dawn advanced the clock, advancing the onset of the Per1 mRNA rhythm and acutely increasing mRNA transcription, whereas light at dusk delayed the clock, delaying the offset of the Per2 mRNA rhythm and tonically increasing mRNA stability. The results suggest that the underlying molecular mechanisms of circadian entrainment differ with morning (advancing) or evening (delaying) light exposure, and such differences may reflect how entrainment takes place in nocturnal animals under natural conditions.

Mechanism of bilateral communication in the suprachiasmatic nucleus.

The central circadian pacemaker of the suprachiasmatic nuclei (SCN) is a bilaterally symmetrical structure. Little is known about the physiological mechanisms underlying communication between the left and right SCN and yet the degree of synchronization between SCN neurons can have a critical impact on the properties of the circadian system. In this study, we used electrophysiological tools and calcium (Ca(2+) ) imaging to examine the mechanisms underlying bilateral signaling in mouse SCN. Electrical stimulation of one SCN produced responses in the contralateral SCN with a short delay (approximately 5 ms) and Ca(2+) -dependence that are consistent with action potential-mediated chemical synaptic transmission. Patch-clamp recordings of stimulated cells revealed excitatory postsynaptic inward-currents (EPSCs), which were sufficient in magnitude to elicit action potentials. Electrical stimulation evoked tetrodotoxin-dependent Ca(2+) transients in about 30% of all contralateral SCN neurons recorded. The responding neurons were widely distributed within the SCN with a highest density in the posterior SCN. EPSCs and Ca(2+) responses were significantly reduced after application of a glutamate receptor antagonist. Application of antagonists for receptors of other candidate transmitters inhibited the Ca(2+) responses in some of the cells but overall the impact of these antagonists was variable. In a functional assay, electrical stimulation of the SCN produced phase shifts in the circadian rhythm in the frequency of multiunit activity rhythm in the contralateral SCN. These phase shifts were blocked by a glutamate receptor antagonist. Taken together, these results implicate glutamate as a transmitter required for communication between the left and right SCN.

Animal clocks: when science meets nature.

Daily rhythms of physiology and behaviour are governed by an endogenous timekeeping mechanism (a circadian 'clock'), with the alternation of environmental light and darkness synchronizing (entraining) these rhythms to the natural day-night cycle. Our knowledge of the circadian system of animals at the molecular, cellular, tissue and organismal levels is remarkable, and we are beginning to understand how each of these levels contributes to the emergent properties and increased complexity of the system as a whole. For the most part, these analyses have been carried out using model organisms in standard laboratory housing, but to begin to understand the adaptive significance of the clock, we must expand our scope to study diverse animal species from different taxonomic groups, showing diverse activity patterns, in their natural environments. The seven papers in this Special Feature of Proceedings of the Royal Society B take on this challenge, reviewing the influences of moonlight, latitudinal clines, evolutionary history, social interactions, specialized temporal niches, annual variation and recently appreciated post-transcriptional molecular mechanisms. The papers emphasize that the complexity and diversity of the natural world represent a powerful experimental resource.

Socially synchronized circadian oscillators.

Daily rhythms of physiology and behaviour are governed by an endogenous timekeeping mechanism (a circadian 'clock'). The alternation of environmental light and darkness synchronizes (entrains) these rhythms to the natural day-night cycle, and underlying mechanisms have been investigated using singly housed animals in the laboratory. But, most species ordinarily would not live out their lives in such seclusion; in their natural habitats, they interact with other individuals, and some live in colonies with highly developed social structures requiring temporal synchronization. Social cues may thus be critical to the adaptive function of the circadian system, but elucidating their role and the responsible mechanisms has proven elusive. Here, we highlight three model systems that are now being applied to understanding the biology of socially synchronized circadian oscillators: the fruitfly, with its powerful array of molecular genetic tools; the honeybee, with its complex natural society and clear division of labour; and, at a different level of biological organization, the rodent suprachiasmatic nucleus, site of the brain's circadian clock, with its network of mutually coupled single-cell oscillators. Analyses at the 'group' level of circadian organization will likely generate a more complex, but ultimately more comprehensive, view of clocks and rhythms and their contribution to fitness in nature.

Beneficial effects of voluntary wheel running on activity rhythms, metabolic state, and affect in a diurnal model of circadian disruption.

Emerging evidence suggests that disruption of circadian rhythmicity contributes to development of comorbid depression, cardiovascular diseases (CVD), and type 2 diabetes mellitus (T2DM). Physical exercise synchronizes the circadian system and has ameliorating effects on the depression- and anxiety-like phenotype induced by circadian disruption in mice and sand rats. We explored the beneficial effects of voluntary wheel running on daily rhythms, and the development of depression, T2DM, and CVD in a diurnal animal model, the fat sand rat (Psammomys obesus). Voluntary exercise strengthened general activity rhythms, improved memory and lowered anxiety- and depressive-like behaviors, enhanced oral glucose tolerance, and decreased plasma insulin levels and liver weight. Animals with access to a running wheel had larger heart weight and heart/body weight ratio, and thicker left ventricular wall. Our results demonstrate that exercising ameliorates pathological-like daily rhythms in activity and blood glucose levels, glucose tolerance and depressive- and anxiety-like behaviors in the sand rat model, supporting the important role of physical activity in modulating the "circadian syndrome" and circadian rhythm-related diseases. We suggest that the utilization of a diurnal rodent animal model may offer an effective way to further explore metabolic, cardiovascular, and affective-like behavioral changes related to chronodisruption and their underlying mechanisms.

A role for the habenula in the regulation of locomotor activity cycles.

Although much is known about the regulation of the circadian rest-activity cycle by the hypothalamic suprachiasmatic nucleus in nocturnal rodents, little is known about the neural substrates that regulate the temporal organization of nocturnal activity within the active phase. In this report, data are presented in Syrian hamsters to implicate the habenula - believed to be involved in motivation, reward and motor control--as a candidate site for such a role. First, by examining hamsters during the day and night and by introducing a 'novel' running wheel in order to induce daytime motor activity, we showed that immunoreactive c-Fos expression in the lateral and medial habenula is related to motor activity/arousal. Second, by transecting the habenula's major efferent pathway (fasciculus retroflexus), we showed that the interruption of habenula neural output alters the daily amount of motor activity, lengthens the period of the circadian rest-activity rhythm and disrupts the species-typical pattern of nocturnal motor activity, measured as either wheel-running behavior or general locomotor activity. Instead of the usual pattern of night-time locomotion, characterized by a prolonged bout of elevated activity in the early night followed by shorter sporadic bouts or the cessation of activity altogether, lesioned animals exhibited a more homogeneous, undifferentiated temporal profile extending across the night. These data suggest a previously unrecognized function of the habenula whereby it regulates the temporal pattern of activity occurring within a circadian rest-activity window set by the suprachiasmatic nucleus.

Embryonic stem cell rescue of tremor and ataxia in myelin-deficient shiverer mice.

Transplantation of neural precursor cells has been proposed as a possible approach for replacing missing or damaged central nervous system myelin. Neonatal and adult myelin-deficient shiverer (shi) mice, bearing a mutation of the myelin basic protein (MBP) gene, have been used extensively as hosts for testing cell engraftment, migration, and myelination, but relatively little progress has been made in reversing shi motor deficits. Here we describe a prenatal cell replacement strategy, showing that embryonic stem cells injected into shi blastocyst embryos can generate chimeric mice with strong and widespread immunoreactive MBP expression throughout the brain and a behavioral (motor) phenotype that appears essentially rescued.

Circadian Neurobiology and the Physiologic Regulation of Sleep and Wakefulness.

Endogenous central and peripheral circadian oscillators are key to organizing multiple aspects of mammalian physiology; this clock tracks the day-night cycle and governs behavioral and physiologic rhythmicity. Flexibility in the timing and duration of sleep and wakefulness, critical to the survival of species, is the result of a complex, dynamic interaction between 2 regulatory processes: the clock and a homeostatic drive that increases with wake duration and decreases during sleep. When circadian rhythmicity and sleep homeostasis are misaligned-as in shifted schedules, time zone transitions, aging, or disease-sleep, metabolic, and other disorders may ensue.

Defective daily temperature regulation in a mouse model of amyotrophic lateral sclerosis.

Current understanding of the pathogenesis of the familial form of amyotrophic lateral sclerosis has been aided by the study of transgenic mice that over-express mutated forms of the human CuZn-superoxide dismutase (SOD1) gene. While mutant SOD1 in motor neurons determines disease onset, other non-cell autonomous factors are critical for disease progression, and altered energy metabolism has been implicated as a contributing factor. Since most energy expended by laboratory mice is utilized to defend body temperature (Tb), we analyzed thermoregulation in transgenic mice carrying the G93A mutation of the human SOD1 gene, using implantable temperature data loggers to continuously record Tb for up to 85 days. At room (22 °C) ambient temperature, G93A mice exhibited a diminished amplitude of the daily Tb rhythm compared to C57BL/6J controls, secondary to decreased Tb values during the dark (behaviorally active) phase of the light-dark cycle. The defect arose at 85-99 days of age, around the age of symptom onset (as assessed by grip strength), well before observable weakness and weight loss, and could not be accounted for by decreased levels of locomotor activity or food consumption. Housing under thermoneutral (29 °C) ambient temperature partially rescued the defect, but age-dependently (only in animals >100 days of age), suggesting that the deficit in older mice was due in part to inadequate thermogenesis by "peripheral" thermogenic organs as the disease progressed. In younger mice, we found that cold-induced thermogenesis and energy expenditure were intact, hinting that an initial "central" defect might localize to the subparaventricular zone, involving neural output pathways from the circadian clock in the hypothalamic suprachiasmatic nucleus to forebrain thermoregulatory circuitry.

Neuronal differentiation of EGF-propagated neurosphere cells after engraftment to the nucleus of the solitary tract.

Neural precursor cells expanded with epidermal growth factor (EGF) exhibit multipotentiality in vitro, but they differentiate predominantly as glial phenotypes after their transplantation in vivo. Here we demonstrate that EGF-propagated precursors from the murine striatal subventricular zone can exhibit robust incorporation and neuronal differentiation within the nucleus of the solitary tract (NST) after injection into the cisterna magna of neonatal or young adult mice. About two-third of engrafted cells appeared NeuN positive in the region of the gelatinous subnucleus, a region notable for its lack of myelinated fibers. The NST may provide a useful model for understanding the physiological and metabolic regulation of postnatal neurogenesis.

Design principles for phase-splitting behaviour of coupled cellular oscillators: clues from hamsters with 'split' circadian rhythms.

Nonlinear interactions among coupled cellular oscillators are likely to underlie a variety of complex rhythmic behaviours. Here we consider the case of one such behaviour, a doubling of rhythm frequency caused by the spontaneous splitting of a population of synchronized oscillators into two subgroups each oscillating in anti-phase (phase-splitting). An example of biological phase-splitting is the frequency doubling of the circadian locomotor rhythm in hamsters housed in constant light, in which the pacemaker in the suprachiasmatic nucleus (SCN) is reconfigured with its left and right halves oscillating in anti-phase. We apply the theory of coupled phase oscillators to show that stable phase-splitting requires the presence of negative coupling terms, through delayed and/or inhibitory interactions. We also find that the inclusion of real biological constraints (that the SCN contains a finite number of non-identical noisy oscillators) implies the existence of an underlying non-uniform network architecture, in which the population of oscillators must interact through at least two types of connections. We propose that a key design principle for the frequency doubling of a population of biological oscillators is inhomogeneity of oscillator coupling.

Modeling the behavior of coupled cellular circadian oscillators in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) in the hypothalamus is the site of the master circadian clock in mammals, a complex tissue composed of multiple, coupled, single-cell circadian oscillators. Mathematical modeling is now providing insights on how individual SCN cells might interact and assemble to create an integrated pacemaker that governs the circadian behavior of whole animals. In this article, we will discuss the neurobiological constraints for modeling SCN behavior, system precision, implications of cellular heterogeneity, and analysis of heterogeneously coupled oscillator networks. Mathematical approaches will be critical for better understanding intercellular interactions within the SCN.

Time management in a co-housed social rodent species (Arvicanthis niloticus).

Sociality has beneficial effects on fitness, and timing the activities of animals may be critical. Social cues could influence daily rhythmic activities via direct effects on the circadian clock or on processes that bypass it (masking), but these possibilities remain incompletely addressed. We investigated the effects of social cues on the circadian body temperature (Tb) rhythms in pairs of co-housed and isolated grass rats, Arvicanthis niloticus (a social species), in constant darkness (DD). Cohabitation did not induce synchronization of circadian Tb rhythms. However, socio-sexual history did affect circadian properties: accelerating the clock in sexually experienced males and females in DD and advancing rhythm phase in the females in a light-dark cycle. To address whether synchronization occurs at an ultradian scale, we analyzed Tb and activity rhythms in pairs of co-housed sisters or couples in DD. Regardless of pair type, co-housing doubled the percentage of time individuals were simultaneously active without increasing individual activity levels, suggesting that activity bouts were synchronized by redistribution over 24 h. Together, our laboratory findings show that social cues affect individual "time allocation" budgets via mechanisms at multiple levels of biological organization. We speculate that in natural settings these effects could be adaptive, especially for group-living animals.

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.