Synchronization-induced rhythmicity of circadian oscillators in the suprachiasmatic nucleus

The suprachiasmatic nuclei (SCN) host a robust, self-sustained circadian pacemaker that coordinates physiological rhythms with the daily changes in the environment. Neuronal clocks within the SCN form a heterogeneous network that must synchronize to maintain timekeeping activity. Coherent circadian output of the SCN tissue is established by intercellular signaling factors, such as vasointestinal polypeptide. It was recently shown that besides coordinating cells, the synchronization factors play a crucial role in the sustenance of intrinsic cellular rhythmicity. Disruption of intercellular signaling abolishes sustained rhythmicity in a majority of neurons and desynchronizes the remaining rhythmic neurons. Based on these observations, the authors propose a model for the synchronization of circadian oscillators that combines intracellular and intercellular dynamics at the single-cell level. The model is a heterogeneous network of circadian neuronal oscillators where individual oscillators are damped rather than self-sustained. The authors simulated different experimental conditions and found that: (1) in normal, constant conditions, coupled circadian oscillators quickly synchronize and produce a coherent output; (2) in large populations, such oscillators either synchronize or gradually lose rhythmicity, but do not run out of phase, demonstrating that rhythmicity and synchrony are codependent; (3) the number of oscillators and connectivity are important for these synchronization properties; (4) slow oscillators have a higher impact on the period in mixed populations; and (5) coupled circadian oscillators can be efficiently entrained by light–dark cycles. Based on these results, it is predicted that: (1) a majority of SCN neurons needs periodic synchronization signal to be rhythmic; (2) a small number of neurons or a low connectivity results in desynchrony; and (3) amplitudes and phases of neurons are negatively correlated. The authors conclude that to understand the orchestration of timekeeping in the SCN, intracellular circadian clocks cannot be isolated from their intercellular communication components.     

Multicellular model for intercellular synchronization in circadian neural networks

We developed a multicellular model characterized by a high degree of heterogeneity to investigate possible mechanisms that underlie circadian network synchronization and rhythmicity in the suprachiasmatic nucleus (SCN). We populated a two-dimensional grid with 400 model neurons coupled via γ-aminobutyric acid (GABA) and vasoactive intestinal polypeptide (VIP) neurotransmitters through a putative Ca²⁺ mediated signaling cascade to investigate their roles in gene expression and electrical firing activity of cell populations. As observed experimentally, our model predicted that GABA would affect the amplitude of circadian oscillations but not synchrony among individual oscillators. Our model recapitulated experimental findings of decreased synchrony and average periods, loss of rhythmicity, and reduced circadian amplitudes as VIP signaling was eliminated. In addition, simulated increases of VIP reduced periodicity and synchrony. We therefore postulated a physiological range of VIP within which the system is able to produce sustained and synchronized oscillations. Our model recapitulated experimental findings of diminished amplitudes and periodicity with decreasing intracellular Ca²⁺ concentrations, suggesting that such behavior could be due to simultaneous decrease of individual oscillation amplitudes and population synchrony. Simulated increases in Cl⁻ levels resulted in increased Cl⁻ influx into the cytosol, a decrease of inhibitory postsynaptic currents, and ultimately a shift of GABA-elicited responses from inhibitory to excitatory. The simultaneous reduction of IPSCs and increase in membrane resting potential produced GABA dose-dependent increases in firing rates across the population, as has been observed experimentally. By integrating circadian gene regulation and electrophysiology with intracellular and intercellular signaling, we were able to develop the first (to our knowledge) multicellular model that allows the effects of clock genes, electrical firing, Ca²⁺, GABA, and VIP on circadian system behavior to be predicted.     

Characteristics of Food-Entrained Orcadian Rhythms in Rats During Long-Term Exposure to Constant Light

Rats possess a system of circadian oscillators that permit entrainment of circadian activity rhythms independently to 24 hr cycles of light-dark and food access. The nature of interactions between food- and light-entrainable oscillators was examined by observing the generation and persistence of food-entrained circadian rhythms in rats whose light-entrainable rhythms were eliminated by long-term exposure to constant light. Most of these rats showed a delayed generation of food-entrained rhythms and only one of eight animals showed persistence of food associated rhythms during a 4-day food deprivation test. Rats whose light-entrainable rhythms are eliminated by suprachiasmatic nuclei ablation show, in contrast, normal generation and persistence of food-entrained rhythms. The results suggested a disruptive influence of constant light on non-photic entrainment, possibly due to coupling forces between damped light-entrainable oscillators and the food-entrainable oscillators.     

Characteristics of food-entrained circadian rhythms in rats during long-term exposure to constant light

Rats possess a system of circadian oscillators that permit entrainment of circadian activity rhythms independently to 24 hr cycles of light-dark and food access. The nature of interactions between food- and light-entrainable oscillators was examined by observing the generation and persistence of food-entrained circadian rhythms in rats whose light-entrainable rhythms were eliminated by long-term exposure to constant light. Most of these rats showed a delayed generation of food-entrained rhythms and only one of eight animals showed persistence of food associated rhythms during a 4-day food deprivation test. Rats whose light-entrainable rhythms are eliminated by suprachiasmatic nuclei ablation show, in contrast, normal generation and persistence of food-entrained rhythms. The results suggested a disruptive influence of constant light on non-photic entrainment, possibly due to coupling forces between damped light-entrainable oscillators and the food-entrainable oscillators.     

A Model for the Fast Synchronous Oscillations of Firing Rate in Rat Suprachiasmatic Nucleus Neurons Cultured in a Multielectrode Array Dish

When dispersed and cultured in a multielectrode dish (MED), suprachiasmatic nucleus (SCN) neurons express fast oscillations of firing rate (FOFR; fast relative to the circadian cycle), with burst duration ∼10 min, and interburst interval varying from 20 to 60 min in different cells but remaining nevertheless rather regular in individual cells. In many cases, separate neurons in distant parts of the 1 mm recording area of a MED exhibited correlated FOFR. Neither the mechanism of FOFR nor the mechanism of their synchronization among neurons is known. Based on recent data implicating vasoactive intestinal polypeptide (VIP) as a key intercellular synchronizing agent, we built a model in which VIP acts as both a feedback regulator to generate FOFR in individual neurons, and a diffusible synchronizing agent to produce coherent electrical output of a neuronal network. In our model, VIP binding to its (VPAC₂) receptors acts through G_s G-proteins to activate adenylyl cyclase (AC), increase intracellular cAMP, and open cyclic-nucleotide-gated (CNG) cation channels, thus depolarizing the cell and generating neuronal firing to release VIP. In parallel, slowly developing homologous desensitization and internalization of VPAC₂ receptors terminates elevation of cAMP and thereby provides an interpulse silent interval. Through mathematical modeling, we show that this VIP/VPAC₂/AC/cAMP/CNG-channel mechanism is sufficient for generating reliable FOFR in single neurons. When our model for FOFR is combined with a published model of synchronization of circadian rhythms based on VIP/VPAC₂ and Per gene regulation synchronization of circadian rhythms is significantly accelerated. These results suggest that (a) auto/paracrine regulation by VIP/VPAC₂ and intracellular AC/cAMP/CNG-channels are sufficient to provide robust FOFR and synchrony among neurons in a heterogeneous network, and (b) this system may also participate in synchronization of circadian rhythms.     

Characteristics of Food-Entrained Orcadian Rhythms in Rats During Long-Term Exposure to Constant Light

Rats possess a system of circadian oscillators that permit entrainment of circadian activity rhythms independently to 24 hr cycles of light-dark and food access. The nature of interactions between food- and light-entrainable oscillators was examined by observing the generation and persistence of food-entrained circadian rhythms in rats whose light-entrainable rhythms were eliminated by long-term exposure to constant light. Most of these rats showed a delayed generation of food-entrained rhythms and only one of eight animals showed persistence of food associated rhythms during a 4-day food deprivation test. Rats whose light-entrainable rhythms are eliminated by suprachiasmatic nuclei ablation show, in contrast, normal generation and persistence of food-entrained rhythms. The results suggested a disruptive influence of constant light on non-photic entrainment, possibly due to coupling forces between damped light-entrainable oscillators and the food-entrainable oscillators.     

Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression

Circadian (ca. 24 hr) oscillations in expression of mammalian "clock genes" are found not only in the suprachiasmatic nucleus (SCN), the central circadian pacemaker, but also in peripheral tissues. Under constant conditions in vitro, however, rhythms of peripheral tissue explants or immortalized cells damp partially or completely. It is unknown whether this reflects an inability of peripheral cells to sustain rhythms, as SCN neurons can, or a loss of synchrony among cells. Using bioluminescence imaging of Rat-1 fibroblasts transfected with a Bmal1::luc plasmid and primary fibroblasts dissociated from mPer2(Luciferase-SV40) knockin mice, we monitored single-cell circadian rhythms of clock gene expression for 1-2 weeks. We found that single fibroblasts can oscillate robustly and independently with undiminished amplitude and diverse circadian periods. Cells were partially synchronized by medium changes at the start of an experiment, but due to different intrinsic periods, their phases became randomly distributed after several days. Closely spaced cells in the same culture did not have similar phases, implying a lack of functional coupling among cells. Thus, like SCN neurons, single fibroblasts can function as independent circadian oscillators; however, lack of oscillator coupling in dissociated cell cultures leads to a loss of synchrony among individual cells and damping of the ensemble rhythm at the population level.     

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.     

Disrupted circadian rhythms in VIP- and PHI-deficient mice

The related neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are expressed at high levels in the neurons of the suprachiasmatic nucleus (SCN), but their function in the regulation of circadian rhythms is unknown. To study the role of these peptides on the circadian system in vivo, a new mouse model was developed in which both VIP and PHI genes were disrupted by homologous recombination. In a light-dark cycle, these mice exhibited diurnal rhythms in activity which were largely indistinguishable from wild-type controls. In constant darkness, the VIP/PHI-deficient mice exhibited pronounced abnormalities in their circadian system. The activity patterns started approximately 8 h earlier than predicted by the previous light cycle. In addition, lack of VIP/PHI led to a shortened free-running period and a loss of the coherence and precision of the circadian locomotor activity rhythm. In about one-quarter of VIP/PHI mice examined, the wheel-running rhythm became arrhythmic after several weeks in constant darkness. Another striking example of these deficits is seen in the split-activity patterns expressed by the mutant mice when they were exposed to a skeleton photoperiod. In addition, the VIP/PHI-deficient mice exhibited deficits in the response of their circadian system to light. Electrophysiological analysis indicates that VIP enhances inhibitory synaptic transmission within the SCN of wild-type and VIP/PHI-deficient mice. Together, the observations suggest that VIP/PHI peptides are critically involved in both the generation of circadian oscillations as well as the normal synchronization of these rhythms to light.     

Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest–activity rhythms and relies upon different groups of PERIOD (PER)–expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day–night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.     

Light Activates Output from Evening Neurons and Inhibits Output from Morning Neurons in the Drosophila Circadian Clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest–activity rhythms and relies upon different groups of PERIOD (PER)–expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day–night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.     

Multiple oscillators in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the pacemaker that controls circadian rhythms of a variety of physiological functions. Data strongly indicate the majority of the SCN neurons express self-sustaining oscillations that can be detected as rhythms in the spontaneous firing of individual neurons. The period of single SCN neurons in a dissociated cell culture is dispersed in a wide range (from 20h to 28h in rats), but that of the locomotor rhythm is close to 24h, suggesting individual oscillators are coupled to generate an averaged circadian period in the nucleus. Electrical coupling via gap junctions, glial regulation, calcium spikes, ephaptic interactions, extracellular ion flux, and diffusible substances have been discussed as possible mechanisms that mediate the interneuronal rhythm synchrony. Recently, GABA (γ-aminobutyric acid), a major neurotransmitter in the SCN, was reported to regulate cellular communication and to synchronize rhythms through GABA_A receptors. At present, subsequent intracellular processes that are able to reset the genetic loop of oscillations are unknown. There may be diverse mechanisms for integrating the multiple circadian oscillators in the SCN. This article reviews the knowledge about the various circadian oscillations intrinsic to the SCN, with particular focus on the intercellular signaling of coupled oscillators. (Chronobiology International, 18(3), 371-387, 2001)     

MULTIPLE OSCILLATORS IN THE SUPRACHIASMATIC NUCLEUS

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the pacemaker that controls circadian rhythms of a variety of physiological functions. Data strongly indicate the majority of the SCN neurons express self-sustaining oscillations that can be detected as rhythms in the spontaneous firing of individual neurons. The period of single SCN neurons in a dissociated cell culture is dispersed in a wide range (from 20h to 28h in rats), but that of the locomotor rhythm is close to 24h, suggesting individual oscillators are coupled to generate an averaged circadian period in the nucleus. Electrical coupling via gap junctions, glial regulation, calcium spikes, ephaptic interactions, extracellular ion flux, and diffusible substances have been discussed as possible mechanisms that mediate the interneuronal rhythm synchrony. Recently, GABA (γ-aminobutyric acid), a major neurotransmitter in the SCN, was reported to regulate cellular communication and to synchronize rhythms through GABA_A receptors. At present, subsequent intracellular processes that are able to reset the genetic loop of oscillations are unknown. There may be diverse mechanisms for integrating the multiple circadian oscillators in the SCN. This article reviews the knowledge about the various circadian oscillations intrinsic to the SCN, with particular focus on the intercellular signaling of coupled oscillators. (Chronobiology International, 18(3), 371–387, 2001)     

Sympathetic input modulates, but does not determine, phase of peripheral circadian oscillators

The circadian clock in the suprachiasmatic nucleus (SCN) maintains phase synchrony among circadian oscillators throughout the organism. Environmental light signals entrain the SCN, but timed, limited meal access acts as an overriding time cue for several peripheral tissues. We present data from a peripheral oscillator, the submaxillary salivary gland, in which temporal restriction of meals fails to entrain gene expression. In day-fed rats, submaxillary gland rhythms in expression of the clock gene Period1 (Per1) stay entrained to the light cycle (peaking at night) or become arrhythmic. This result suggests that feeding cues compete weakly with light cycle cues to set the phase of clock genes in this tissue. Since the submaxillary glands receive sympathetic innervation originating in the SCN, which relays light cycle cues to other oscillators, we attempted to assess the role of this neural input in phase control of submaxillary Per1 expression. We sympathetically denervated the submaxillary glands before subjecting rats to daytime-restricted feeding. After denervation, Per1 rhythms in all submaxillary glands shifted phase 180 degrees and entrained to daytime feeding. These results support the hypothesis that peripheral oscillators may receive multiple signals contributing to their phase of entrainment. Sympathetic efferents from the SCN can relay light cycle information, while other external cues may reach tissues through other efferents or nonneural pathways. In an abnormal, disruptive regimen such as daytime-restricted feeding, these different signals compete. Arrhythmicity may result if one signal is not clearly dominant. Elimination of the dominant signal (e.g., surgical sympathectomy) may allow a secondary signal to control phase.