Mammalian Biological Clock Research Articles

CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9

BackgroundClock circadian regulator (CLOCK) is a core factor of the mammalian biological clock system in regulating female fertility and ovarian physiology. However, CLOCK's specific function and molecular mechanism in porcine granulosa cells (GCs) remain unclear. In this study, we focused on CLOCK’s effects on GC proliferation.ResultsCLOCK significantly inhibited cell proliferation in porcine GCs. CLOCK decreased the expression of cell cycle-related genes, including CCNB1, CCNE1, and CDK4 at the mRNA and protein levels. CDKN1A levels were upregulated by CLOCK. ASB9 is a newly-identified target of CLOCK that inhibits GC proliferation; CLOCK binds to the E-box element in the ASB9 promoter.ConclusionsThese findings suggest that CLOCK inhibits the proliferation of porcine ovarian GCs by increasing ASB9 level.

Distinct contribution of cone photoreceptor subtypes to the mammalian biological clock

Ambient light detection is important for the synchronization of the circadian clock to the external solar cycle. Light signals are sent to the suprachiasmatic nuclei (SCN), the site of the major circadian pacemaker. It has been assumed that cone photoreceptors contribute minimally to synchronization. Here, however, we find that cone photoreceptors are sufficient for mediating entrainment and transmitting photic information to the SCN, as evaluated in mice that have only cones as functional photoreceptors. Using in vivo electrophysiological recordings in the SCN of freely moving cone-only mice, we observed light responses in SCN neuronal activity in response to 60-s pulses of both ultraviolet (UV) (λmax 365 nm) and green (λmax 505 nm) light. Higher irradiances of UV light led to irradiance-dependent enhancements in SCN neuronal activity, whereas higher irradiances of green light led to a reduction in the sustained response with only the transient response remaining. Responses in SCN neuronal activity decayed with a half-max time of ∼9 min for UV light and less than a minute for green light, indicating differential input between short-wavelength-sensitive and mid-wavelength-sensitive cones for the SCN responsiveness. Furthermore, we show that UV light is more effective for photoentrainment than green light. Based on the lack of a full sustained response in cone-only mice, we confirmed that rapidly alternating light levels, rather than slowly alternating light, caused substantial phase shifts. Together, our data provide strong evidence that cone types contribute to photoentrainment and differentially affect the electrical activity levels of the SCN.

Imaging Individual Differences in the Response of the Human Suprachiasmatic Area to Light.

Circadian disruption is associated with poor health outcomes, including sleep and mood disorders. The suprachiasmatic nucleus (SCN) of the anterior hypothalamus acts as the master biological clock in mammals, regulating circadian rhythms throughout the body. The clock is synchronized to the day/night cycle via retinal light exposure. The BOLD-fMRI response of the human suprachiasmatic area to light has been shown to be greater in the night than in the day, consistent with the known sensitivity of the clock to light at night. Whether the BOLD-fMRI response of the human suprachiasmatic area to light is related to a functional outcome has not been demonstrated. In a pilot study (n = 10), we investigated suprachiasmatic area activation in response to light in a 30 s block-paradigm of lights on (100 lux) and lights off (< 1 lux) using the BOLD-fMRI response, compared to each participant's melatonin suppression response to moderate indoor light (100 lux). We found a significant correlation between activation in the suprachiasmatic area in response to light in the scanner and melatonin suppression, with increased melatonin suppression being associated with increased suprachiasmatic area activation in response to the same light level. These preliminary findings are a first step toward using imaging techniques to measure individual differences in circadian light sensitivity, a measure that may have clinical relevance in understanding vulnerability in disorders that are influenced by circadian disruption.

Enkephalin and neuropeptide-Y interaction in the intergeniculate leaflet network, a part of the mammalian biological clock

The intergeniculate leaflet (IGL) is a flat thalamic nucleus implicated in the modulation of circadian rhythmicity. In rat, two main GABAergic subpopulations can be distinguished in the IGL: neurons synthesizing neuropeptide Y (NPY), which directly innervates the suprachiasmatic nuclei, and enkephalinergic cells, which connect contralaterally located leaflets. The aim of this study was to evaluate possible effects of inner IGL neurotransmitters on the spontaneous and synaptic activity of IGL neurons. The data presented in this article provide evidence that enkephalin, and not NPY, could act upon the majority of IGL neurons. Moreover, we investigated the type of opioid receptor activated by enkephalin and showed that the μ-receptor is functionally predominant in the IGL. The application of met-enkephalin not only robustly hyperpolarized IGL neurons (both putatively NPY-synthesizing and putatively enkephalinergic neurons), but it also was able to inhibit GABAergic and glutamatergic synaptic transmission. Based on this and previous studies, we hypothesize that IGL enkephalinergic neurons may act as powerful interneurons that inhibit themselves and NPY-synthesizing neurons, also in the contralaterally located IGL.

Orexins/hypocretins modulate the activity of NPY-positive and -negative neurons in the rat intergeniculate leaflet via OX1 and OX2 receptors

Orexins/hypocretins (OXA and OXB) are two hypothalamic peptides involved in the regulation of many physiological processes including the sleep-wake cycle, food intake and arousal. The orexinergic system of the lateral hypothalamus is considered a non-specific peptidergic system, and its nerve fibers innervate numerous brain areas. Among many targets of orexinergic neurons is the intergeniculate leaflet (IGL) of the thalamus – a small but important structure of the mammalian biological clock. In rats, the IGL consists of GABAergic cells which also synthesize different neuropeptides. One group of neurons produces neuropeptide Y (NPY) and sends its axons to the master biological clock known as the suprachiasmatic nuclei. Another neuronal group produces enkephalin and is known to connect contralateral IGLs. This study evaluated the effects of orexins on identified IGL neurons revealing that 58% of the recorded neurons were sensitive to OXA (200nM) and OXB (200nM) administration. Both NPY-positive and -negative neurons were depolarized by these neuropeptides. Experiments using selective orexin receptor antagonists (SB-334867, 10μM and TCS-OX2-29, 10μM) suggested that both orexin receptors participate in the recorded OXA effects. In addition, IGL neurons were either directly depolarized by OXA or their activity was altered by changes in presynaptic inputs. We observed an increase of GABA release onto the investigated IGL neuron after OXA application, consistent with a presynaptic localization of the orexin receptors. An increase in miniature excitatory postsynaptic current frequency was not observed within the IGL. Our findings reinforce the connection between circadian clock physiology and the orexinergic system.

N‐nitrosomelatonin enhances photic synchronization of mammalian circadian rhythms

Most physiological processes in mammals are synchronized to the daily light:dark cycle by a circadian clock located in the hypothalamic suprachiasmatic nucleus. Signal transduction of light-induced phase advances of the clock is mediated through a neuronal nitric oxide synthase-guanilyl cyclase pathway. We have employed a novel nitric oxide-donor, N-nitrosomelatonin, to enhance the photic synchronization of circadian rhythms in hamsters. The intraperitoneal administration of this drug before a sub-saturating light pulse at circadian time 18 generated a twofold increase of locomotor rhythm phase-advances, having no effect over saturating light pulses. This potentiation was also obtained even when inhibiting suprachiasmatic nitric oxide synthase activity. However, N-nitrosomelatonin had no effect on light-induced phase delays at circadian time 14. The photic-enhancing effects were correlated with an increased suprachiasmatic immunoreactivity of FBJ murine osteosarcoma viral oncogene and period1. Moreover, in vivo nitric oxide release by N-nitrosomelatonin was verified by measuring nitrate and nitrite levels in suprachiasmatic nuclei homogenates. The compound also accelerated resynchronization to an abrupt 6-h advance in the light:dark cycle (but not resynchronization to a 6-h delay). Here, we demonstrate the chronobiotic properties of N-nitrosomelatonin, emphasizing the importance of nitric oxide-mediated transduction for circadian phase advances.

Intrinsic organization of the suprachiasmatic nucleus in the capuchin monkey

The suprachiasmatic nucleus (SCN), which is the main circadian biological clock in mammals, is composed of multiple cells that function individually as independent oscillators to express the self-sustained mRNA and protein rhythms of the so-called clock genes. Knowledge regarding the presence and localization of the proteins and neuroactive substances of the SCN are essential for understanding this nucleus and for its successful manipulation. Although there have been advances in the investigation of the intrinsic organization of the SCN in rodents, little information is available in diurnal species, especially in primates. This study, which explores the pattern of expression and localization of PER2 protein in the SCN of capuchin monkey, evaluates aspects of the circadian system that are common to both primates and rodents. Here, we showed that PER2 protein immunoreactivity is higher during the light phase. Additionally, the complex organization of cells that express vasopressin, vasoactive intestinal polypeptide, neuron-specific nuclear protein, calbindin and calretinin in the SCN, as demonstrated by their immunoreactivity, reveals an intricate network that may be related to the similarities and differences reported between rodents and primates in the literature.

Light and chronobiology: implications for health and disease

Environmental light synchronizes the primary mammalian biological clock in the suprachiasmatic nuclei, as well as many peripheral clocks in tissues and cells, to the solar 24-hour day. Light is the strongest synchronizing agent (zeitgeber) for the circadian system, and therefore keeps most biological and psychological rhythms internally synchronized, which is important for optimum function. Circadian sleep-wake disruptions and chronic circadian misalignment, as often observed in psychiatric and neurodegenerative illness, can be treated with light therapy. The beneficial effect on circadian synchronization, sleep quality, mood, and cognitive performance depends on timing, intensity, and spectral composition of light exposure. Tailoring and optimizing indoor lighting conditions may be an approach to improve wellbeing, alertness, and cognitive performance and, in the long term, producing health benefits.

De rol van de biologische klok en het autonome zenuwstelsel bij wakker worden

Background Awakening from sleep is a clear example of an event for which the biological clock is of great importance. We will review some major pathways the mammalian biological clock uses to ensure an efficient and coordinated wake-up process.

Mammalian clock output mechanisms

In mammals many behaviours (e.g. sleep-wake, feeding) as well as physiological (e.g. body temperature, blood pressure) and endocrine (e.g. plasma corticosterone concentration) events display a 24 h rhythmicity. These 24 h rhythms are induced by a timing system that is composed of central and peripheral clocks. The highly co-ordinated output of the hypothalamic biological clock not only controls the daily rhythm in sleep-wake (or feeding-fasting) behaviour, but also exerts a direct control over many aspects of hormone release and energy metabolism. First, we present the anatomical connections used by the mammalian biological clock to enforce its endogenous rhythmicity on the rest of the body, especially the neuro-endocrine and energy homoeostatic systems. Subsequently, we review a number of physiological experiments investigating the functional significance of this neuro-anatomical substrate. Together, this overview of experimental data reveals a highly specialized organization of connections between the hypothalamic pacemaker and neuro-endocrine system as well as the pre-sympathetic and pre-parasympathetic branches of the autonomic nervous system.

Vasopressin and the Output of the Hypothalamic Biological Clock

The physiological effects of vasopressin as a peripheral hormone were first reported more than 100 years ago. However, it was not until the first immunocytochemical studies were carried out in the early 1970s, using vasopressin antibodies, and the discovery of an extensive distribution of vasopressin-containing fibres outside the hypothalamus, that a neurotransmitter role for vasopressin could be hypothesised. These studies revealed four additional vasopressin systems next to the classical magnocellular vasopressin system in the paraventricular and supraoptic nuclei: a sexually dimorphic system originating from the bed nucleus of the stria terminalis and the medial amygdala, an autonomic and endocrine system originating from the medial part of the paraventricular nucleus, and the circadian system originating from the hypothalamic suprachiasmatic nuclei (SCN). At about the same time as the discovery of the neurotransmitter function of vasopressin, it also became clear that the SCN contain the main component of the mammalian biological clock system (i.e. the endogenous pacemaker). This review will concentrate on the significance of the vasopressin neurones in the SCN for the functional output of the biological clock that is contained within it. The vasopressin-containing subpopulation is a characteristic feature of the SCN in many species, including humans. The activity of the vasopressin neurones in the SCN shows a pronounced daily variation in its activity that has also been demonstrated in human post-mortem brains. Animal experiments show an important role for SCN-derived vasopressin in the control of neuroendocrine day/night rhythms such as that of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes. The remarkable correlation between a diminished presence of vasopressin in the SCN and a deterioration of sleep-wake rhythms during ageing and depression make it likely that, also in humans, the vasopressin neurones contribute considerably to the rhythmic output of the SCN.

Circadian Control of the Daily Plasma Glucose Rhythm: An Interplay of GABA and Glutamate

The mammalian biological clock, located in the hypothalamic suprachiasmatic nuclei (SCN), imposes its temporal structure on the organism via neural and endocrine outputs. To further investigate SCN control of the autonomic nervous system we focused in the present study on the daily rhythm in plasma glucose concentrations. The hypothalamic paraventricular nucleus (PVN) is an important target area of biological clock output and harbors the pre-autonomic neurons that control peripheral sympathetic and parasympathetic activity. Using local administration of GABA and glutamate receptor (ant)agonists in the PVN at different times of the light/dark-cycle we investigated whether daily changes in the activity of autonomic nervous system contribute to the control of plasma glucose and plasma insulin concentrations. Activation of neuronal activity in the PVN of non-feeding animals, either by administering a glutamatergic agonist or a GABAergic antagonist, induced hyperglycemia. The effect of the GABA-antagonist was time dependent, causing increased plasma glucose concentrations only when administered during the light period. The absence of a hyperglycemic effect of the GABA-antagonist in SCN-ablated animals provided further evidence for a daily change in GABAergic input from the SCN to the PVN. On the other hand, feeding-induced plasma glucose and insulin responses were suppressed by inhibition of PVN neuronal activity only during the dark period. These results indicate that the pre-autonomic neurons in the PVN are controlled by an interplay of inhibitory and excitatory inputs. Liver-dedicated sympathetic pre-autonomic neurons (responsible for hepatic glucose production) and pancreas-dedicated pre-autonomic parasympathetic neurons (responsible for insulin release) are controlled by inhibitory GABAergic contacts that are mainly active during the light period. Both sympathetic and parasympathetic pre-autonomic PVN neurons also receive excitatory inputs, either from the biological clock (sympathetic pre-autonomic neurons) or from non-clock areas (para-sympathetic pre-autonomic neurons), but the timing information is mainly provided by the GABAergic outputs of the biological clock.

Opposite actions of hypothalamic vasopressin on circadian corticosterone rhythm in nocturnal versus diurnal species

Relatively little is known about the function of the biological clock and its efferent pathways in diurnal species, despite the fact that its major transmitters and neuronal connections are also conserved in humans. The mammalian biological clock is located in the hypothalamic suprachiasmatic nuclei (SCN). Several lines of evidence suggest that the activity cycle of the SCN itself is similar in nocturnal and diurnal mammals. Previously, we showed that, in the rat, vasopressin (VP) derived from the SCN has a strong inhibitory effect on the release of adrenal corticosterone and is an important component in the generation of a daily rhythm in plasma corticosterone concentrations. In the present study we investigated the role of VP in the control of the daily corticosterone rhythm in a diurnal rodent, i.e. Arvicanthis ansorgei. Contrary to our previous (rat) results, VP administered to the hypothalamic paraventricular nucleus in A. ansorgei had a stimulatory effect on the release of corticosterone. Moreover, both the morning and evening rise in corticosterone were blocked by the administration of a VP receptor antagonist. These results show that with regard to the circadian control of the corticosterone rhythm in diurnal and nocturnal rodents, temporal information is carried along the same pathway from the SCN to its target areas, but the response of the target area may be quite different. We propose that the reversed response to VP is due to a change in the phenotype of the target neurons that are contacted by the SCN efferents, i.e. glutamatergic instead of gamma-aminobutyric acid (GABA)ergic.

Minireview: Circadian Control of Metabolism by the Suprachiasmatic Nuclei

In the present review, first we present the anatomical connections used by the mammalian biological clock to enforce its endogenous rhythmicity on the rest of the body, especially the energy homeostatic systems. Subsequently, we present a number of physiological experiments investigating the functional significance of this neuroanatomical substrate. Together, this overview of experimental data, for a major part derived from our own experiments, reveals a highly specialized organization of connections between the endogenous pacemaker and both the presympathetic and pre-parasympathetic hypothalamic systems, providing the biological clock with a unique opportunity to modulate the balance of sympathetic/parasympathetic inputs to peripheral organs. We hypothesize that a well-balanced autonomic nervous input, differentiated according to the time of day and the body compartment, is an important companion to withstand the progressive burden of the current 24/7 society on our health and well-being.

Clock genes beyond the clock: CLOCK genotype biases neural correlates of moral valence decision in depressed patients

Gene polymorphisms in the mammalian biological clock system influence individual rhythms. A single nucleotide polymorphism (SNP) in the 3' flanking region of CLOCK (3111 T/C; rs1801260) influenced diurnal preference in healthy humans and caused sleep phase delay and insomnia in patients affected by bipolar disorder. Genes of the biological clock are expressed in many brain structures other than in the 'master clock' suprachiasmatic nuclei. These areas, such as cingulate cortex, are involved in the control of many human behaviors. Clock genes could then bias 'nonclock' functions such as information processing and decision making. Thirty inpatients affected by a major depressive episode underwent blood oxygen-level dependent (BOLD) functional magnetic resonance imaging (fMRI). The cognitive activation paradigm was based on a go/no-go task. Morally connoted words were presented. Genotyping of CLOCK was performed for each patients. We measured activity levels through actimetry during the day before the fMRI study. CLOCK 3111 T/C SNP was associated with activity levels in the second part of the day, neuropsychological performance and BOLD fMRI correlates (interaction of genotype and moral valence of the stimuli). Our results support the hypothesis that individual clock genotype may influence several variables linked with human behaviors in normal and psychopathological conditions.

Effects of physical exercise on human circadian rhythms

Bright light is the principal zeitgeber for the biological clock in mammals, including humans. But there is a line of evidence that non-photic stimuli such as physical activity play an important role in entrainment. Scheduled physical activity, such as wheel and forced treadmill running, has been reported to phase-shift and entrain the circadian rhythm in rodent species. In humans, several studies have reported the phase-shifting effects of physical exercise. A single bout of physical exercise at night was demonstrated to phase-delay the circadian rhythm in plasma melatonin. However, for the entrainment of human circadian rhythm, a phase-advance shift is needed. Previously, we demonstrated that scheduled physical exercise in the waking period facilitated the entrainment of plasma melatonin rhythm to the sleep/wake schedule of 23 h 40 min. This result suggested that timed physical exercise produced phase-advance shifts. A regular physical exercise also facilitated entrainment of the circadian rhythms associated with acute phase-delay shifts of the sleep/wake and light/dark schedule. These findings suggest that physical exercise is useful to adjust the circadian rhythm to external time cues, especially for totally blind people and elderly people.

A novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus

The suprachiasmatic nucleus of the anterior hypothalamus is the center of an internal biological clock in mammals. Glutamate is the neurotransmitter of retino-hypothalamic tract responsible for mediating the circadian actions of light in rodents. N-methyl- d-aspartate receptors, particularly NR2B subunit are reported to be principally involved in photic resetting of the biological clock in vivo and in slice culture. But, the precise cellular mechanisms of the resetting are not elucidated, because no adequate neuronal cell lines derived from the suprachiasmatic nucleus have been established. We established a neuronal cell line, N14.5, derived from the suprachiasmatic nucleus of a transgenic rat harboring the temperature-sensitive simian virus 40 large T-antigen gene. When the cells were cultured at 39 °C, the morphological features were turned fibroblastic into neuronal round cell body with neurite extensions. These cells showed immunoreactivities for neuronal markers ( βIII-tubulin, microtubule-associated protein 2 and TAU2) and as well as for vasoactive intestinal peptide which is expressed in the ventrolateral region of the suprachiasmatic nucleus. The cells expressed N-methyl- d-aspartate receptors, particularly NR1 and NR2B subunits as revealed by quantitative PCR. N-methyl- d-aspartate activated phosphorylation of p44/42 mitogen-activated protein kinase and increased expression level of Per1 and Per2 mRNA. These results suggest that the N14.5 is a novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus, and that N-methyl- d-aspartate receptors expressed in the cells are a functional receptor. The N14.5 cells may be a useful tool to elucidate numerous chronobiological processes, especially resetting mechanism induced by an external light signal.

Dynamical Heterogeneity of Suprachiasmatic Nucleus Neurons Based on Regularity and Determinism

The suprachiasmatic nucleus (SCN) is known to be the master biological clock in mammals. Despite the periodic mean firing rate, interspike interval (ISI) patterns of SCN neurons are quite complex and irregular. The aim of the present study was to investigate the existence of nonlinear determinism in the complex ISI patterns of SCN neurons. ISI sequences were recorded from 173 neurons in rat hypothalamic slice preparations using a cell-attached patch recording technique. Their correlation dimensions (D2) were estimated, and were then compared with those of the randomly-shuffled surrogate data. We found that only 16 neurons (16/173) exhibited deterministic ISI patterns of spikes. In addition, clustering analysis revealed that SCN neurons could be divided into two subgroups of neurons each having distinct values of coefficient of variation (CV) and skewness (SK). Interestingly, most deterministic SCN neurons (14/16) belonged to the group of irregularly spiking neurons having large CV and SK values. To see if the neuronal coupling mediated by the gamma-aminobutyric acid (GABA), the major neurotransmitter in the SCN, contributed to the deterministic nature, we examined the effect of the GABAA receptor antagonist bicuculline on D2 values of 56 SCN neurons. 8 SCN neurons which were originally stochastic became to exhibit deterministic characteristics after the bicuculline application. This result suggests that the deterministic nature of the SCN neurons arises not from GABAergic synaptic interactions, but likely from properties inherent to neurons themselves.

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 (approximately 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 GABAA 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.

Tissue‐specific expression of tryptophan hydroxylase mRNAs in the rat midbrain: anatomical evidence and daily profiles

Serotonin (5-HT) is involved in both photic and non-photic synchronization of the mammalian biological clock located in the suprachiasmatic nuclei (SCN). We have previously demonstrated that tryptophan hydroxylase protein (TPH), the rate-limiting enzyme of 5-HT synthesis, shows circadian rhythmicity in the pathways projecting from the raphe nuclei to the intergeniculate leaflets of the thalamus on one hand, and to the SCN on the other hand. In this study, we investigate whether the circadian rhythmicity in TPH protein could result from the rhythmic expression of tph gene in the raphe nuclei. We thus cloned specific tph1 and tph2 partial cDNAs and assessed the daily profiles of TPH mRNA levels by in situ hybridization in the rat raphe nuclei. Our results demonstrate that: (i) tph2 gene is exclusively expressed in the raphe nuclei, whereas tph1 gene is expressed in the pineal gland; (ii) under light-dark cycle (LD), TPH2 mRNA levels present daily variation within both median and dorsal raphe nuclei; (iii) under constant darkness TPH2 mRNA levels in both nuclei exhibit the same variation reported under LD cycle. These data show that the circadian 5-HT synthesis within the serotonergic neurons projecting to the circadian system might be explained by the rhythmic transcription of the tph2 gene in raphe nuclei. Taking our result with previous data into consideration, we further suggest that 5-HT synthesis and release within the circadian system could be directly or indirectly under the control of the SCN.