Cellular communication and coupling within the suprachiasmatic nucleus

In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a pair of structures in the hypothalamus known as the suprachiasmatic nucleus (SCN). Importantly, when SCN neurons are removed from the organism and maintained in a brain slice preparation, they continue to generate 24h rhythms in electrical activity, secretion, and gene expression. Previous studies suggest that the basic mechanism responsible for the generation of these rhythms is intrinsic to individual cells in the SCN. If we assume that individual cells in the SCN are competent circadian oscillators, it is obviously important to understand how these cells communicate and remain synchronized with each other. Cell-to-cell communication is clearly necessary for conveying inputs to and outputs from the SCN and may be involved in ensuring the high precision of the observed rhythm. In addition, there is a growing body of evidence that a number of systems-level phenomena could be dependent on the cellular communication between circadian pacemaker neurons. It is not yet known how this cellular synchronization occurs, but it is likely that more than one of the already proposed mechanisms is utilized. The purpose of this review is to summarize briefly the possible mechanisms by which the oscillatory cells in the SCN communicate with each other. (Chronobiology International, 18(4)579-600, 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)     

Come together, right...now: synchronization of rhythms in a mammalian circadian clock

In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus act as a dominant circadian pacemaker, coordinating rhythms throughout the body and regulating daily and seasonal changes in physiology and behavior. This review focuses on the mechanisms that mediate synchronization of circadian rhythms between SCN neurons. Understanding how these neurons communicate as a network of circadian oscillators has begun to shed light on the adaptability and dysfunction of the brain's master clock.     

Serotonergic innervation of the hypothalamic suprachiasmatic nucleus and photic regulation of circadian rhythms

Converging lines of evidence have firmly established that the hypothalamic suprachiasmatic nucleus (SCN) is a light-entrainable circadian oscillator in mammals, critically important for the expression of behavioral and physiological circadian rhythms. Photic information essential for the daily phase resetting of the SCN circadian clock is conveyed directly to the SCN from retinal ganglion cells via the retinohypothalamic tract. The SCN also receives a dense serotonergic innervation arising from the mesencephalic raphe. The terminal fields of retinal and serotonergic afferents within the SCN are co-extensive, and serotonergic agonists can modify the response of the SCN circadian oscillator to light. However, the functional organization and subcellular localization of 5HT receptor subtypes in the SCN are just beginning to be clarified. This information is necessary to understand the role 5HT afferents play in modulating photic input to the SCN. In this paper, we review evidence suggesting that the serotonergic modulation of retinohypothalamic neurotransmission may be achieved via at least two different cellular mechanisms: 1) a postsynaptic mechanism mediated via 5HT_1A or 5ht₇ receptors located on SCN neurons; and 2) a presynaptic mechanism mediated via 5HT_1B receptors located on retinal axon terminals in the SCN. Activation of either of these 5HT receptor mechanisms in the SCN by specific 5HT agonists inhibits the effects of light on circadian function. We hypothesize that 5HT modulation of photic input to the SCN may serve to set the gain of the SCN circadian system to light.     

Electrophysiology of the Circadian Pacemaker in Mammals

The neurons of the mammalian suprachiasmatic nuclei (SCN) control circadian rhythms in molecular, physiological, endocrine, and behavioral functions. In the SCN, circadian rhythms are generated at the level of individual neurons. The last decade has provided a wealth of information on the genetic basis for circadian rhythm generation. In comparison, a modest but growing number of studies have investigated how the molecular rhythm is translated into neuronal function. Neuronal attributes have been measured at the cellular and tissue level with a variety of electrophysiological techniques. We have summarized electrophysiological research on neurons that constitute the SCN in an attempt to provide a comprehensive view on the current state of the art.     

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)     

Electrophysiology of the circadian pacemaker in mammals

The neurons of the mammalian suprachiasmatic nuclei (SCN) control circadian rhythms in molecular, physiological, endocrine, and behavioral functions. In the SCN, circadian rhythms are generated at the level of individual neurons. The last decade has provided a wealth of information on the genetic basis for circadian rhythm generation. In comparison, a modest but growing number of studies have investigated how the molecular rhythm is translated into neuronal function. Neuronal attributes have been measured at the cellular and tissue level with a variety of electrophysiological techniques. We have summarized electrophysiological research on neurons that constitute the SCN in an attempt to provide a comprehensive view on the current state of the art.     

Modeling the spontaneous activity in suprachiasmatic nucleus neurons: Role of cation single channels

A population of interconnected neurons of the mammalian suprachiasmatic nuclei (SCN) controls circadian rhythms in physiological functions. In turn, a circadian rhythm of individual neurons is driven by intracellular processes, which via activation of specific membrane channels, produce circadian modulation of electrical firing rate. Yet the membrane target(s) of the cellular clock have remained enigmatic. Previously, subthreshold voltage-dependent cation (SVC) channels have been proposed as the membrane target of the cellular clock responsible for circadian modulation of the firing rate in SCN neurons. We tested this hypothesis with computational modeling based on experimental results from on-cell recording of SVC channel openings in acutely isolated SCN neurons and long-term continuous recording of activity from dispersed SCN neurons in a multielectrode array dish (MED). The model reproduced the circadian behavior if the number of SVC channels or their kinetics were modulated in accordance with protein concentration in a model of the intracellular clock (Scheper et al., 1999. J. Neurosci. 19, 40-47). Such modulation changed the average firing rate of the model neuron from zero (“subjective-night” silence) up to 18 Hz (“subjective-day” peak). Furthermore, the variability of interspike intervals (ISI) and the circadian pattern of firing rate (i.e. silence-to-activity ratio and shape of circadian peaks) are in reasonable agreement with experimental data obtained in dispersed SCN neurons in MED. These results suggest that the variability of ISI in intact SCN neurons is mostly due to stochastic single-channel openings, and that the circadian pattern of the firing rate is specified by threshold properties of dependence of the spontaneous firing rate on the number of single channels (R-N relationship). This plausible mathematical modeling supports the hypothesis that SVC channels could be a critical element in circadian modulation of firing rate in SCN neurons.     

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.     

Clock genes, oscillators, and cellular networks in the suprachiasmatic nuclei

The mammalian SCN contains a biological clock that drives remarkably precise circadian rhythms in vivo and in vitro. Recent advances have revealed molecular and cellular mechanisms required for the generation of these daily rhythms and their synchronization between SCN neurons and to the environmental light cycle. This review of the evidence for a cell-autonomous circadian pacemaker within specialized neurons of the SCN focuses on 6 genes implicated within the pace making mechanism, an additional 4 genes implicated in pathways from the pacemaker, and the intercellular and intracellular mechanisms that synchronize SCN neurons to each other and to solar time.     

Rhythmic coupling among cells in the suprachiasmatic nucleus

In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a pair of structures in the hypothalamus known as the suprachiasmatic nucleus (SCN). Previous studies suggest that the basic mechanism responsible for the generation of these rhythms is intrinsic to individual cells. There is also evidence that the cells within the SCN are coupled to one another and that this coupling is important for the normal functioning of the circadian system. One mechanism that mediates coordinated electrical activity is direct electrical connections between cells formed by gap junctions. In the present study, we used a brain slice preparation to show that developing SCN cells are dye coupled. Dye coupling was observed in both the ventrolateral and dorsomedial subdivisions of the SCN and was blocked by application of a gap junction inhibitor, halothane. Dye coupling in the SCN appears to be regulated by activity-dependent mechanisms as both tetrodotoxin and the GABA(A) agonist muscimol inhibited the extent of coupling. Furthermore, acute hyperpolarization of the membrane potential of the original biocytin-filled neuron decreased the extent of coupling. SCN cells were extensively dye coupled during the day when the cells exhibit synchronous neural activity but were minimally dye coupled during the night when the cells are electrically silent. Immunocytochemical analysis provides evidence that a gap-junction-forming protein, connexin32, is expressed in the SCN of postnatal animals. Together the results are consistent with a model in which gap junctions provide a means to couple SCN neurons on a circadian basis.     

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.     

Coupling-induced synchronization in multicellular circadian oscillators of mammals

In mammals, circadian rhythms are controlled by the neurons located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Each neuron in the SCN contains an autonomous molecular clock. The fundamental question is how the individual cellular oscillators, expressing a wide range of periods, interact and assemble to achieve phase synchronization. Most of the studies carried out so far emphasize the crucial role of the periodicity imposed by the light-dark cycle in neuronal synchronization. However, in natural conditions, the interaction between the SCN neurons is non-negligible and coupling between cells in the SCN is achieved partly by neurotransmitters. In this paper, we use a model of nonidentical, globally coupled cellular clocks considered as Goodwin oscillators. We mainly study the synchronization induced by coupling from an analytical way. Our results show that the role of the coupling is to enhance the synchronization to the external forcing. The conclusion of this paper can help us better understand the mechanism of circadian rhythm.     

mPer2 antisense oligonucleotides inhibit mPER2 expression but not circadian rhythms of physiological activity in cultured suprachiasmatic nucleus neurons

Various day-night rhythms, observed at molecular, cellular, and behavioral levels, are governed by an endogenous circadian clock, predominantly functioning in the hypothalamic suprachiasmatic nucleus (SCN). A class of clock genes, mammalian Period (mPer), is known to be rhythmically expressed in SCN neurons, but the correlation between mPER protein levels and autonomous rhythmic activity in SCN neurons is not well understood. Therefore, we blocked mPer translation using antisense phosphothioate oligonucleotides (ODNs) for mPer1 and mPer2 mRNAs and examined the effects on the circadian rhythm of cytosolic Ca2+ concentration and action potentials in SCN slice cultures. Treatment with mPer2 ODNs (20microM for 3 days) but not randomized control ODNs significantly reduced mPER2 immunoreactivity (-63%) in the SCN. Nevertheless, mPer1/2 ODNs treatment inhibited neither action potential firing rhythms nor cytosolic Ca2+ rhythms. These suggest that circadian rhythms in mPER protein levels are not necessarily coupled to autonomous rhythmic activity in SCN neurons.     

The cell adhesion molecule EphA4 is involved in circadian clock functions

Circadian (～24 h) rhythms of cellular network plasticity in the central circadian clock, the suprachiasmatic nucleus (SCN), have been described. The neuronal network in the SCN regulates photic resetting of the circadian clock as well as stability of the circadian system during both entrained and constant conditions. EphA4, a cell adhesion molecule regulating synaptic plasticity by controlling connections of neurons and astrocytes, is expressed in the SCN. To address whether EphA4 plays a role in circadian photoreception and influences the neuronal network of the SCN, we have analyzed circadian wheel‐running behavior of EphA4 knockout (EphA4^?/?) mice under different light conditions and upon photic resetting, as well as their light‐induced protein response in the SCN. EphA4^?/? mice exhibited reduced wheel‐running activity, longer endogenous periods under constant darkness and shorter periods under constant light conditions, suggesting an effect of EphA4 on SCN function. Moreover, EphA4^?/? mice exhibited suppressed phase delays of their wheel‐running activity following a light pulse during the beginning of the subjective night (CT15). Accordingly, light‐induced c‐FOS (FBJ murine osteosarcoma viral oncogene homolog) expression was diminished. Our results suggest a circadian role for EphA4 in the SCN neuronal network, affecting the circadian system and contributing to the circadian response to light.     

Intercellular coupling confers robustness against mutations in the SCN circadian clock network

Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.