Characterization of Proteases in the Hamster Suprachiasmatic Nucleus

The activities of several proteases in the hamster suprachiasmatic nuclei were measured at different time points throughout the daily or circadian cycle. No variation for metalloproteinase A (MMP-2) activity was found, while metalloproteinase B (MMP-9) was rhythmic and maximally active during the night. In addition, diurnal variations for two low molecular weight proteases were determined, with peaks during the light phase. This rhythmicity appears to be under exogenous control, since constant darkness abolished fluctuations throughout the circadian cycle. These results suggest that protein degradation in the hamster circadian clock is regulated in a diurnal fashion.     

Constructing Suprachiasmatic Nucleus Chimeras In Vivo

We introduce a new transplantation technique for analyzing suprachiasmatic nucleus (SCN) development and function. Neural precursor ('stem') cells are harvested from the brains of mice engineered to express the green fluorescent protein, propagated in culture with growth factors, and injected into the forebrain ventricles of individual embryos in order to gain access to the ventricular germinal zone and the developing brain. Initial data indicate that such cells can incorporate within the SCN and appear to differentiate into astroglial phenotypes. In addition to applications in chronobiology, SCN chimeras may be a potentially powerful model system for analyzing the structural and functional plasticity of implanted precursor cells.     

Constructing Suprachiasmatic Nucleus Chimeras In Vivo

We introduce a new transplantation technique for analyzing suprachiasmatic nucleus (SCN) development and function. Neural precursor (‘stem’) cells are harvested from the brains of mice engineered to express the green fluorescent protein, propagated in culture with growth factors, and injected into the forebrain ventricles of individual embryos in order to gain access to the ventricular germinal zone and the developing brain. Initial data indicate that such cells can incorporate within the SCN and appear to differentiate into astroglial phenotypes. In addition to applications in chronobiology, SCN chimeras may be a potentially powerful model system for analyzing the structural and functional plasticity of implanted precursor cells.     

大鼠下丘脑交叉上核中CREB的昼夜节律

利用凝胶迁移率变化的实验方法,对饲养在光照－黑暗循环的条件和持续黑暗的条件下Wistar雄性大鼠下丘脑交叉上核中CREB含量的昼夜间变化进行了分析,发现CREB在交叉上核中具有内源性的昼夜节律.     

哺乳动物核移植中供核与受体卵胞质细胞周期的相互关系

就供核与受体卵胞质细胞周期的相互关系问题进行了综述.核移植技术不管是在基础理论,还是在应用研究中都具有广泛的应用价值,但核移植的效率却很低,其根本原因是与核移植相关的许多基础理论问题尚不清楚,对这些问题的研究发现,维持重构卵核的正确倍性,并使其重新程序化是核移植成功的关键,不同的胞质受体及不同的供体细胞及其状态均对重构胚的发育有影响.     

Nucleus replacement in Mammalian oocytes

Our contribution discusses the potential use of cell therapies (nucleus replacement) in mammalian oocytes. It is assumed that these approaches may be used, for example, for the elimination of mutated maternally transmitted mitochondrial DNA (mtDNA) as well as for the reconstruction of normal oocytes from oocytes that are developmentally compromised. Moreover, it is speculated that the replacement of germinal vesicles by somatic cells may result in cells of the haploid genome: the production of germ cells from somatic cells. The preliminary results obtained in our laboratories are discussed in this article.     

The Suprachiasmatic Nucleus is not Required for Temporal Gating of Performance on a Reward-based Learning and Memory Task

In hamsters, the expression of a learned preference for context depends upon a temporal match between the time of training and testing. In the present experiments, we investigated the role of the biological clock in the suprachiasmatic nucleus (SCN) as a provider of temporal information underlying this time dependent modulation of cognitive performance. Hamsters were tested using the conditioned place preference task (CPP) before and after ablation of the SCN. Arrhythmic animals continued to show time-of-day modulation of the CPP when trained and tested in the absence of the SCN. This supports the notion that time of day information is a component of context representation for the hamster (Antoniadis et al., 1999), and indicates that an oscillator outside of the SCN is responsible for time discrimination in reward-based learning.     

Fractal Stochastic Modeling of Spiking Activity in Suprachiasmatic Nucleus Neurons

Individual neurons in the suprachiasmatic nucleus (SCN), the master biological clock in mammals, autonomously produce highly complex patterns of spikes. We have shown that most (~90%) SCN neurons exhibit truly stochastic interspike interval (ISI) patterns. The aim of this study was to understand the stochastic nature of the firing patterns in SCN neurons by analyzing the ISI sequences of 150 SCN neurons in hypothalamic slices. Fractal analysis, using the periodogram, Fano factor, and Allan factor, revealed the presence of a 1/f-type power-law (fractal) behavior in the ISI sequences. This fractal nature was persistent after the application of the GABA_A receptor antagonist bicuculline, suggesting that the fractal stochastic activity is an intrinsic property of individual SCN neurons. Based on these physiological findings, we developed a computational model for the stochastic SCN neurons to find that their stochastic spiking activity was best described by a gamma point process whose mean firing rate was modulated by a fractal binomial noise. Taken together, we suggest that SCN neurons generate temporal spiking patterns using the fractal stochastic point process.Action Editor: Carson C. Chow     

The Role of the Suprachiasmatic Nucleus in Cardiac Autonomic Control during Sleep

Background

The suprachiasmatic nucleus (SCN) may play an important role in central autonomic control, since its projections connect to (para)sympathetic relay stations in the brainstem and spinal cord. The cardiac autonomic modifications during nighttime may therefore not only result from direct effects of the sleep-related changes in the central autonomic network, but also from endogenous circadian factors as directed by the SCN. To explore the influence of the SCN on autonomic fluctuations during nighttime, we studied heart rate and its variability (HRV) in a clinical model of SCN damage.

Methods

Fifteen patients in follow-up after surgical treatment for nonfunctioning pituitary macroadenoma (NFMA) compressing the optic chiasm (8 females, 26–65 years old) and fifteen age-matched healthy controls (5 females, 30–63 years) underwent overnight ambulatory polysomnography. Eleven patients had hypopituitarism and received adequate replacement therapy. HRV was calculated for each 30-second epoch and corrected for sleep stage, arousals, and gender using mixed effect regression models.

Results

Compared to controls, patients spent more time awake after sleep onset and in NREM1-sleep, and less in REM-sleep. Heart rate, low (LF) and high frequency (HF) power components and the LF/HF ratio across sleep stages were not significantly different between groups.

Conclusions

These findings suggest that the SCN does not play a dominant role in cardiac autonomic control during sleep.     

Efferent Projections of Prokineticin 2 Expressing Neurons in the Mouse Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) in the hypothalamus is the predominant circadian clock in mammals. To function as a pacemaker, the intrinsic timing signal from the SCN must be transmitted to different brain regions. Prokineticin 2 (PK2) is one of the candidate output molecules from the SCN. In this study, we investigated the efferent projections of PK2-expressing neurons in the SCN through a transgenic reporter approach. Using a bacterial artificial chromosome (BAC) transgenic mouse line, in which the enhanced green fluorescence protein (EGFP) reporter gene expression was driven by the PK2 promoter, we were able to obtain an efferent projections map from the EGFP-expressing neurons in the SCN. Our data revealed that EGFP-expressing neurons in the SCN, hence representing some of the PK2-expressing neurons, projected to many known SCN target areas, including the ventral lateral septum, medial preoptic area, subparaventricular zone, paraventricular nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamic area and paraventricular thalamic nucleus. The efferent projections of PK2-expressing neurons supported the role of PK2 as an output molecule of the SCN.     

Entrainment of the Human Circadian Clock to the Natural Light-Dark Cycle

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Characterization of Melatonin-Induced FOS-Like Immunoreactivity in the Hypothalamic Suprachiasmatic Nucleus of the Rat

Abstract

The hypothalamic suprachiasmatic nucleus (SCN) is primarily responsible for the regulation of circadian rhythmicity. Melatonin, the pineal-derived neurohormone, modulates the rhythmic output of the SCN. Properly timed exposure to melatonin is able to induce changes in rhythrnic function and thereby entrain circadian rhythms of activity.

c-fos is an immediate early gene that is transiently expressed in neurons in response to receptor activation. The ventrolateral portion of the SCN (vSCN) is activated in response to phase-shifting stimuli, an event which is marked by an increase in the expression of c-fos.

In the present study, rats systemically administered the melatonin agonist 2-iodomelatonin at CT 22 demonstrated significant dose-dependent Fos immunoreactivity within the vSCN, an effect which was significantly inhibited by the melatonin antagonist N-acetyltryptamine. The Fos expression observed in the vSCN was not affected by treatment with the serotonin antagonist ketanserin or the alpha-adrenergic antagonist phentolamine. Moreover, antisense oligonucleotides to c-fos, significantly blocked the ability of 2-iodomelatonin to induce Fos expression in the vSCN at CT 22.

These results pharmacologically characterize melatonin-induced c-fos expression in the rat vSCN and provide evidence to support a c-fos-mediated mechanism through which the activation of melatonin receptors may be linked to the long-term molecular regulation of circadian rhythms controlled by the SCN.     

Temporal Morphology and Cap Formation in Acetabulars-I. Light-Dark Cycle Perturbations

La formation du chapeau, qui constitue un événement morphogénétique de la plus grande importance chez ?'algue Acetabularïa, est influénce par une perturbation unique du cycle lumière-obscurité, done aussi, vraisemblablement, des rythmes circadiens. La perturbation a lieu plusieurs semaines avant ?expression morphogénétique. ? effet est plus marqué sur la formation du chapeau que sur la croissance. Son importance dépend du moment du nyctémère où la perturbation intervient.

?effet dépend du stade de développement de ?'algue: lorsque le transfert d' un régime L-D à un autre a lieu pendant la phase de grande croissance, la formation du chapeau est retardée, mais ? importance de ? effet s'atténue avec le temps. Lorsque le transfert a lieu pendant la phase terminale de croissance (lente), la formation du chapeau est accélérée de façon transitoire. Lorsqu'il a lieu alors que les algues ont presque atteint leur taille finale, il est sans effet. Ces résultats présentent des analogies avec le photopériodisme.

Mots clefs–Acetabularia, rythmes circadiens, morphogénèse, photopériodisme.

Cap formation, a major developmental process in the alga Acetabularia, is influenced by a single perturbation of the entraining light-dark schedule and thus, presumably, of the circadian rhythms. This perturbation is brought about several weeks before cap formation, the most conspicuous expression of morphogenesis in Acetabularia. The effect is more pronounced on cap formation than on growth. It varies in importance with the circadian time at which the perturbation was brought about.

The effect is dependent on the developmental state of the alga: transfer carried out during the logarithmic phase of growth produces a delay whose importance decreases with time. When carried out during the phase of slow terminal growth, the transfer induces a transistory acceleration of cap formation. When the algae approach their final length, no effect is elicited. Photoperiodism seems to be involved.     

Differential Involvement of the Suprachiasmatic Nucleus in Lipopolysaccharide-Induced Plasma Glucose and Corticosterone Responses

The hypothalamic suprachiasmatic nucleus (SCN) is an essential component of the circadian timing system, and an important determinant of neuroendocrine and metabolic regulation. Recent data indicate a modulatory role for the immune system on the circadian timing system. The authors investigated how the circadian timing system affects the hypothalamo-pituitary-adrenal (HPA) axis and glucose regulatory responses evoked by an immune challenge induced by lipopolysaccharide (LPS). LPS-induced increases in corticosterone were minimal during the trough of the daily corticosterone rhythm; in contrast, LPS effects on glucose, glucagon, and insulin did not vary across time-of-day. Complete ablation of the SCN resulted in increased corticosterone responses but did not affect LPS-induced hyperglycemia. The paraventricular nucleus (PVN) of the hypothalamus is an important neuroendocrine and autonomic output pathway for hypothalamic information, as well as one of the main target areas of the SCN. Silencing the neuronal activity in the PVN did not affect the LPS-induced corticosterone surge and only slightly delayed the LPS-induced plasma glucose and glucagon responses. Finally, surgical interruption of the neuronal connection between hypothalamus and liver did not affect the corticosterone response but slightly delayed the LPS-induced glucose response. Together, these data support the previously proposed circadian modulation of LPS-induced neuroendocrine responses, but they are at variance with the suggested major role for the hypothalamic pacemaker on the autonomic output of the hypothalamus, as reflected by the effects of LPS on glucose homeostasis. The latter effects are more likely due to direct interactions of LPS with peripheral tissues, such as the liver. (Author correspondence: a.kalsbeek@amc.uva.nl)     

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca²⁺ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.