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Find video protocols related to scientific articles indexed in Pubmed.
New methods to assess circadian clocks in humans.
Indian J. Exp. Biol.
PUBLISHED: 05-24-2014
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Proper function of the circadian system seems crucial for human health. New advances in methods for assessment of the functional state of the human circadian system facilitate our understanding of the relationship between the disruption of the circadian system and various diseases. Based on the results of such studies, new directions for the diagnosis and treatment of diseases emerge. This communication aims to summarize current methods for evaluating the human circadian system in the laboratory as well as in field studies. The advantages and limitations of the current methods and various approaches used for both in vivo and in vitro assessment of the human circadian system are discussed.
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Melatonin administered during the fetal stage affects circadian clock in the suprachiasmatic nucleus but not in the liver.
Dev Neurobiol
PUBLISHED: 05-14-2014
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The mammalian circadian system develops gradually during ontogenesis, and after birth, the system is already set to a phase of the mothers. The role of maternal melatonin in the entrainment of fetal circadian clocks has been suggested, but direct evidence is lacking. In our study, intact or pinealectomized pregnant rats were exposed to constant light (LL) throughout pregnancy to suppress the endogenous melatonin and behavioral rhythms. During the last 5 days of gestation, the rats were injected with melatonin or vehicle or were left untreated. After delivery, daily expression profiles of c-fos and Avp in the suprachiasmatic nuclei (SCN), and Per1, Per2, Rev-erb?, and Bmal1 in the liver were measured in 1-day-old pups. Due to the LL exposure, no gene expression rhythms were detected in the SCN of untreated pregnant rats or in the SCN and liver of the pups. The administration of melatonin to pregnant rats entrained the pups' gene expression profiles in the SCN, but not in the liver. Melatonin did not affect the maternal behavior during pregnancy. Vehicle injections also synchronized the gene expression in the SCN but not in the liver. Melatonin and vehicle entrained the gene expression profiles to different phases, demonstrating that the effect of melatonin was apparently not due to the treatment procedure per se. The data demonstrate that in pregnant rats with suppressed endogenous melatonin levels, pharmacological doses of melatonin affect the fetal clock in the SCN but not in the liver. © 2014 Wiley Periodicals, Inc. Develop Neurobiol, 2014.
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Cross-talk between the circadian clock and the cell cycle in cancer.
Ann. Med.
PUBLISHED: 04-30-2014
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The circadian clock is an endogenous timekeeper system that controls the daily rhythms of a variety of physiological processes. Accumulating evidence indicates that genetic changes or unhealthy lifestyle can lead to a disruption of circadian homeostasis, which is a risk factor for severe dysfunctions and pathologies including cancer. Cell cycle, proliferation, and cell death are closely intertwined with the circadian clock, and thus disruption of circadian rhythms appears to be linked to cancer development and progression. At the molecular level, the cell cycle machinery and the circadian clocks are controlled by similar mechanisms, including feedback loops of genes and protein products that display periodic activation and repression. Here, we review the circadian rhythmicity of genes associated with the cell cycle, proliferation, and apoptosis, and we highlight the potential connection between these processes, the circadian clock, and neoplastic transformations. Understanding these interconnections might have potential implications for the prevention and therapy of malignant diseases.
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The circadian system of patients with bipolar disorder differs in episodes of mania and depression.
Bipolar Disord
PUBLISHED: 03-04-2014
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Bipolar disorder is a common psychiatric disease characterized by mood disturbances with alternating episodes of mania and depression. Moreover, disturbances in the sleep/wake cycle are prevalent. We tested a hypothesis that the function of the circadian system, which drives the sleep/wake cycle, may differ in patients with bipolar disorder depending on whether they are experiencing an episode of mania or depression.
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In vivo initiation of clock gene expression rhythmicity in fetal rat suprachiasmatic nuclei.
PLoS ONE
PUBLISHED: 01-01-2014
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The mammalian suprachiasmatic nuclei (SCN) and their intrinsic rhythmicity develop gradually during ontogenesis. In the rat, the SCN forms between embryonic day (E) 14 and E17, with gestation terminating at E21-22. Overt SCN rhythmicity is already present in the late embryonic stage. The aim of the present study was to determine when the fetal SCN clock develops in vivo and whether overt rhythmicity results from a functional fetal clock. To achieve this goal, the prenatal development of rhythmic expression of clock genes was measured with a more sensitive method for detection of the clock gene expression than previously. Fetal SCN were collected at 3 h intervals during the 24 h period on E19 and E21 by laser dissection and expression of clock genes (Per2, Nr1d1 and Bmal1) and genes related to cellular activity (c-fos, Avp and Vip) was measured by qRT PCR. At E19, the expression of canonical clock genes Per2 and Bmal1 was not rhythmic; however, the expression of all other studied genes followed clear circadian rhythms. At E21, Per2 and Bmal1 expression exhibited low amplitude but significant rhythmicity. From E19 to E21, the levels of the non-rhythmic transcripts (Per2 and Bmal1) decreased; however, the levels of the rhythmic transcripts (Nr1d1, c-fos, Avp and Vip) increased. In summary, these data demonstrate that at E19, rhythms in Per2 and Bmal1 expression were absent in the fetal SCN; however, the expression of Nr1d1 and other genes related to cellular activity was driven rhythmically. Therefore, at the early stage in vivo, the developing fetal SCN clock could theoretically be entrained by oscillation of Nr1d1 which may be driven by the maternal rather than fetal circadian system.
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Nogo-A-deficient Transgenic Rats Show Deficits in Higher Cognitive Functions, Decreased Anxiety, and Altered Circadian Activity Patterns.
Front Behav Neurosci
PUBLISHED: 01-01-2014
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Decreased levels of Nogo-A-dependent signaling have been shown to affect behavior and cognitive functions. In Nogo-A knockout and knockdown laboratory rodents, behavioral alterations were observed, possibly corresponding with human neuropsychiatric diseases of neurodevelopmental origin, particularly schizophrenia. This study offers further insight into behavioral manifestations of Nogo-A knockdown in laboratory rats, focusing on spatial and non-spatial cognition, anxiety levels, circadian rhythmicity, and activity patterns. Demonstrated is an impairment of cognitive functions and behavioral flexibility in a spatial active avoidance task, while non-spatial memory in a step-through avoidance task was spared. No signs of anhedonia, typical for schizophrenic patients, were observed in the animals. Some measures indicated lower anxiety levels in the Nogo-A-deficient group. Circadian rhythmicity in locomotor activity was preserved in the Nogo-A knockout rats and their circadian period (tau) did not differ from controls. However, daily activity patterns were slightly altered in the knockdown animals. We conclude that a reduction of Nogo-A levels induces changes in CNS development, manifested as subtle alterations in cognitive functions, emotionality, and activity patterns.
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Development and entrainment of the colonic circadian clock during ontogenesis.
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 12-12-2013
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Colonic morphology and function change significantly during ontogenesis. In mammals, many colonic physiological functions are temporally controlled by the circadian clock in the colon, which is entrained by the central circadian clock in the suprachiasmatic nuclei (SCN). The aim of this present study was to ascertain when and how the circadian clock in the colon develops during the perinatal period and whether maternal cues and/or the developing pup SCN may influence the ontogenesis of the colonic clock. Daily profiles of clock genes Per1, Per2, Cry1, Cry2, Rev-erb?, Bmal1 and Clock expression in the colon underwent significant modifications since embryonic day 20 (E20) through postnatal days (P) 2, 10, 20 and 30 via changes in the mutual phasing among the individual clock gene expression rhythms, their relative phasing to the light/dark regime, and their amplitudes. An adult-like state was achieved around P20. The foster study revealed that during the prenatal period, the maternal circadian phase may partially modulate development of the colonic clock. Postnatally, the absence and/or presence of rhythmic maternal care affected the phasing of the clock gene expression profiles in pups at P10 and P20. A reversal in the colonic clock phase between P10 and P20 occurred in absence of rhythmic signals from the pup SCN. The data demonstrate ontogenetic maturation of the colonic clock and stress the importance of prenatal and postnatal maternal rhythmic signals for its development. These data may contribute to the understanding of colonic function-related diseases in newborn children.
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Human chronotype is determined in bodily cells under real-life conditions.
Chronobiol. Int.
PUBLISHED: 02-27-2013
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Individuals differ in their preferred timing of sleep and activity, which is referred to as a chronotype. The timing shows a wide distribution; extremely early chronotypes may wake up when the extremely late chronotypes fall asleep. The chronotype is supposed to be determined by the central circadian clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus because the phasing of the pineal melatonin rhythm, which is driven by the SCN, correlates with the sleep timing preference. In addition to the SCN, circadian oscillators are also present in most if not all bodily cells. These peripheral clocks are synchronized by the central SCN clock and by other tissue-specific entraining cues. At the molecular level, the circadian oscillations are based on a complex, self-sustaining mechanism that drives the rhythmical expression of clock genes and their proteins. The aim of the present field study was to elucidate whether the changes in the internal timing of early and late chronotypes, as expressed by changes in the phases of their mid-sleep and melatonin secretion, can also be detected at the molecular clockwork level in subjects examined under real-life conditions. Ninety-five adult volunteers were chronotyped using an adapted Munich chronotype questionnaire to assess their mid-sleep phase, and 6 subjects with early chronotypes and 6 with late chronotypes were chosen for the study. For the assessment of the circadian phase, the subjects provided samples of saliva for the melatonin assay and samples of oral mucosa for the determination of clock gene Per1, Per2, and Rev-erb? mRNA levels every 4?h during a 24-h period. The significant correlation between the phase of the melatonin profile and timing of mid-sleep confirmed the classification of the subjects according to their chronotype. The circadian phases of the Per1, Per2, and Rev-erb? expression profiles in the oral mucosa were advanced in the early chronotypes compared with those in the late chronotypes (p < .001) and correlated significantly with the mid-sleep phase of the individual subjects. Moreover, the circadian phases of the Per1 expression profiles of individual subjects correlated significantly with the phases of their melatonin profiles (p < .05), whereas the correlation for the Per2 and Rev-erb? phases was nonsignificant, although the trend was the same. Our results demonstrate that the individual chronotype in humans living in real-life conditions affects not only the phasing of the daily melatonin rhythm in saliva but also the phasing of Per1, Per2, and Rev-erb? clock gene expression profiles in buccal mucosa cells. This report represents the first demonstration that the human peripheral circadian clock may sense the individuals chronotype under field study conditions. The data contribute to our understanding of the mechanisms underlying human chronotypes in real life.
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Entrainment of spontaneously hypertensive rat fibroblasts by temperature cycles.
PLoS ONE
PUBLISHED: 01-01-2013
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The functional state of the circadian system of spontaneously hypertensive rats (SHR) differs in several characteristics from the functional state of normotensive Wistar rats. Some of these changes might be due to the compromised ability of the central pacemaker to entrain the peripheral clocks. Daily body temperature cycles represent one of the important cues responsible for the integrity of the circadian system, because these cycles are driven by the central pacemaker and are able to entrain the peripheral clocks. This study tested the hypothesis that the aberrant peripheral clock entrainment of SHR results from a compromised peripheral clock sensitivity to the daily temperature cycle resetting. Using cultured Wistar rat and SHR fibroblasts transfected with the circadian luminescence reporter Bmal1-dLuc, we demonstrated that two consecutive square-wave temperature cycles with amplitudes of 2.5 °C are necessary and sufficient to restart the dampened oscillations and entrain the circadian clocks in both Wistar rat and SHR fibroblasts. We also generated a phase response curve to temperature cycles for fibroblasts of both rat strains. Although some of the data suggested a slight resistance of SHR fibroblasts to temperature entrainment, we concluded that the overall effect it too weak to be responsible for the differences between the SHR and Wistar in vivo circadian phenotype.
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Increased sensitivity of the circadian system to temporal changes in the feeding regime of spontaneously hypertensive rats - a potential role for bmal2 in the liver.
PLoS ONE
PUBLISHED: 01-01-2013
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The mammalian timekeeping system generates circadian oscillations that rhythmically drive various functions in the body, including metabolic processes. In the liver, circadian clocks may respond both to actual feeding conditions and to the metabolic state. The temporal restriction of food availability to improper times of day (restricted feeding, RF) leads to the development of food anticipatory activity (FAA) and resets the hepatic clock accordingly. The aim of this study was to assess this response in a rat strain exhibiting complex pathophysiological symptoms involving spontaneous hypertension, an abnormal metabolic state and changes in the circadian system, i.e., in spontaneously hypertensive rats (SHR). The results revealed that SHR were more sensitive to RF compared with control rats, developing earlier and more pronounced FAA. Whereas in control rats, the RF only redistributed the activity profiles into two bouts (one corresponding to FAA and the other corresponding to the dark phase), in SHR the RF completely phase-advanced the locomotor activity according to the time of food presentation. The higher behavioral sensitivity to RF was correlated with larger phase advances of the hepatic clock in response to RF in SHR. Moreover, in contrast to the controls, RF did not suppress the amplitude of the hepatic clock oscillation in SHR. In the colon, no significant differences in response to RF between the two rat strains were detected. The results suggested the possible involvement of the Bmal2 gene in the higher sensitivity of the hepatic clock to RF in SHR because, in contrast to the Wistar rats, the rhythm of Bmal2 expression was advanced similarly to that of Bmal1 under RF. Altogether, the data demonstrate a higher behavioral and circadian responsiveness to RF in the rat strain with a cardiovascular and metabolic pathology and suggest a likely functional role for the Bmal2 gene within the circadian clock.
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Alteration of the circadian clock in children with Smith-Magenis syndrome.
J. Clin. Endocrinol. Metab.
PUBLISHED: 12-07-2011
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Smith-Magenis syndrome (SMS) is associated with sleep disturbances and disrupted melatonin production.
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Salivary melatonin rhythm as a marker of the circadian system in healthy children and those with attention-deficit/hyperactivity disorder.
Chronobiol. Int.
PUBLISHED: 08-24-2011
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Attention-deficit/hyperactivity disorder (ADHD) is the most common neurobehavioral disorder of childhood. Problems with sleep structure, efficiency, and timing have been reported in some, but not all, studies on ADHD children. As the sleep-wake cycle belongs to circadian rhythms, the timekeeping circadian system might be involved in ADHD. To assess whether the circadian system of ADHD children differs from that of controls, the rhythm of the pineal hormone melatonin was used as a reliable marker of the system. Saliva from 34 ADHD and 43 control 6- to 12-yr-old children was sampled at 2-h intervals throughout the entire 24-h cycle, and the melatonin profiles of the ADHD and control children were compared. The nocturnal melatonin peaks of the ADHD and control group did not differ significantly. The high nocturnal interindividual variability of the peaks seen in adulthood was present already in the studied children. The 24-h melatonin profiles of all the ADHD subjects did not differ significantly from those of the control subjects. Categorization of subjects according to age, into groups of 6- to 7-yr-old (9 ADHD, 5 control), 8- to 9-yr-old (16 ADHD, 26 control), and 10- to 12-yr-old (9 ADHD, 12 control) children, revealed significant differences between the ADHD and control group in the melatonin rhythm waveform, but not in nocturnal melatonin peaks; the peaks were about the same in both groups and did not change significantly with increasing age. In the oldest, but not in the younger, children, the melatonin signal duration in the ADHD group was shorter than in the control group. The difference might be due to the fact that whereas in the control group both the evening melatonin onset and the morning offset phase delayed in the oldest children relative to those in the youngest children, in the ADHD group only the onset, but not the offset, phase delayed with increasing age. The data may indicate subtle differences between the circadian system of ADHD and control children during development.
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Hepatic, duodenal, and colonic circadian clocks differ in their persistence under conditions of constant light and in their entrainment by restricted feeding.
Chronobiol. Int.
PUBLISHED: 04-02-2011
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Physiological functions of the gastrointestinal tract (GIT) are temporally controlled such that they exhibit circadian rhythms. The circadian rhythms are synchronized with the environmental light-dark cycle via signaling from the central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus, and by food intake. The aim of the study was to determine the extent to which disturbance in the SCN signaling via prolonged exposure to constant light affects circadian rhythms in the liver, duodenum, and colon, as well as to determine whether and to what extent food intake can restore rhythmicity in individual parts of the GIT. Adult male rats were maintained in constant light (LL) for 30 days and fed ad libitum throughout the entire interval or exposed to a restricted feeding (RF) regime for the last 14 days in LL. Locomotor and feeding behaviors were recorded throughout the experiment. On the 30th day, daily expression profiles of clock genes (Per1, Per2, Rev-erb?, and Bmal1) and of clock-controlled genes (Wee1 and Dbp) were measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in the duodenum, colon, and liver. By the end of the LL exposure, rats fed ad libitum had completely lost their circadian rhythms in activity and food intake. Daily expression profiles of clock genes and clock-controlled genes in the GIT were impaired to an extent depending on the tissue and gene studied, but not completely abolished. In the liver and colon, exposure to LL abolished circadian rhythms in expression of Per1, Per2, Bmal1, and Wee1, whereas it impaired, but preserved, rhythms in expression of Rev-erb? and Dbp. In the duodenum, all but Wee1 expression rhythms were preserved. Restricted feeding restored the rhythms to a degree that varied with the tissue and gene studied. Whereas in the liver and duodenum the profiles of all clock genes and clock-controlled genes became rhythmic, in the colon only Per1, Bmal1, and Rev-erb?-but not Per2, Wee1, and Dbp-were expressed rhythmically. The data demonstrate a greater persistence of the rhythmicity of the circadian clocks in the duodenum compared with that in the liver and colon under conditions when signaling from the SCN is disrupted. Moreover, disrupted rhythmicity may be restored more effectively by a feeding regime in the duodenum and liver compared to the colon.
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Exposure of pregnant rats to restricted feeding schedule synchronizes the SCN clocks of their fetuses under constant light but not under a light-dark regime.
J. Biol. Rhythms
PUBLISHED: 09-30-2010
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The circadian clock in the suprachiasmatic nucleus (SCN) develops gradually during the prenatal and early postnatal period. In the rat, this period lasts from around the 15th day of gestation until the 10th day of postnatal development. The circadian system of fetuses and newborn pups is entrained mostly by nonphotic maternal cues during prenatal and early postnatal development. The aim of the present study was to ascertain whether exposure of pregnant rats to a restricted feeding (RF) regime was able to entrain the circadian clock in the SCN of their fetuses during the prenatal period. The potency of RF as an entraining cue was tested under conditions when pregnant rats were entrained to an external light/dark (LD) cycle as well as under conditions when the external timing signal was lacking, i.e., under constant light (LL). The control groups were fed ad libitum and the experimental groups had restricted access to food for 6 h during their resting time throughout pregnancy. Daily profiles of Avp and c-fos gene expression were examined by in situ hybridization in the SCN of 1-day-old pups. The data demonstrated that RF in pregnant rats kept under LD cycle did not notably affect the daily rhythms of c-fos and Avp expression in the SCN of pups. The SCN profiles of Avp and c-fos gene expression did not exhibit circadian rhythms in pups born to mothers maintained in LL and fed ad libitum, likely due to desynchrony among the pups within a litter. However, RF in the pregnant rats kept under LL restored the circadian rhythmicity of c-fos and Avp expression in the SCN of their newborn pups. The results suggest that the fetal SCN clock is dominantly entrained by rhythmic signals from the maternal SCN. However, under conditions when the rhythmic signaling might be lacking, such as LL, regular food intake of the mothers may also play an important role in synchronization of the fetal SCN clock during prenatal ontogenesis.
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Expression and light sensitivity of clock genes Per1 and Per2 and immediate-early gene c-fos within the retina of early postnatal Wistar rats.
J. Comp. Neurol.
PUBLISHED: 07-01-2010
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Mammalian retina contains a circadian clock that is composed of components similar to those of the master circadian clock within the suprachiasmatic nuclei of the hypothalamus. The aim of the present study was to elucidate whether, when, and where the transcripts of the clock genes Per1 and Per2 and the immediate early gene c-fos are spontaneously expressed and/or induced by light in the newborn rat retina. At postnatal day 1 (P1), P3, P5, and P10, Wistar rat pups were released into constant darkness, and a 30-minute light pulse was administered during the subjective day or during the first or second part of subjective night. Gene expression was determined 30 minutes, 1 hour, 2 hours, and 4 hours after the light pulse by in situ hybridization followed by emulsion autoradiography. Endogenous expression of Per1 was detected in the neuroblastic retina, and Per2 expression was detected in the inner part of the neuroblastic retina from birth. Light pulses induced c-fos expression in ganglion cells from P1. Until P5, the cells were localized in the dorsal part of the retina, but, at P10, they were already distributed across the entire retinal circumference. Light pulses also induced the expression of c-fos and Per1 in the retinal pigment epithelium until P3, but not afterward. Expression of the Per2 gene was not photoresponsive until P10. These data demonstrate that the rat retina is light-sensitive immediately after birth. During early postnatal development, the spatial distribution of spontaneous and light-induced gene expression within the retinal layers changes gradually.
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Different mechanisms of adjustment to a change of the photoperiod in the suprachiasmatic and liver circadian clocks.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
PUBLISHED: 01-13-2010
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Changes in photoperiod modulate the circadian system, affecting the function of the central clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The aim of the present study was to elucidate the dynamics of adjustment to a change of a long photoperiod with 18 h of light to a short photoperiod with 6 h of light of clock gene expression rhythms in the mouse SCN and in the peripheral clock in the liver, as well as of the locomotor activity rhythm. Three, five, and thirteen days after the photoperiod change, daily profiles of Per1, Per2, and Rev-erbalpha expression in the rostral, middle, and caudal parts of the SCN and of Per2 and Rev-erbalpha in the liver were determined by in situ hybridization and real-time RT-PCR, respectively. The clock gene expression rhythms in the different SCN regions, desynchronized under the long photoperiod, attained synchrony gradually following the transition from long to short days, mostly via advancing the expression decline. The photoperiodic modulation of the SCN was due not only to the degree of synchrony among the SCN regions but also to different waveforms of the rhythms in the individual SCN parts. The locomotor activity rhythm adjusted gradually to short days by advancing the activity onset, and the liver rhythms adjusted by advancing the Rev-erbalpha expression rise and Per2 decline. These data indicate different mechanisms of adjustment to a change of the photoperiod in the central SCN clock and the peripheral liver clock.
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Influence of photoperiod duration and light-dark transitions on entrainment of Per1 and Per2 gene and protein expression in subdivisions of the mouse suprachiasmatic nucleus.
Eur. J. Neurosci.
PUBLISHED: 10-14-2009
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The circadian clock located within the suprachiasmatic nuclei (SCN) of the hypothalamus responds to changes in the duration of day length, i.e. photoperiod. Recently, changes in phase relationships among the SCN cell subpopulations, especially between the rostral and caudal region, were implicated in the SCN photoperiodic modulation. To date, the effect of abrupt, rectangular, light-to-dark transitions have been studied while in nature organisms experience gradual dawn and twilight transitions. The aim of this study was to compare the effect of a long (18 h of light) and a short (6 h of light) photoperiod with twilight relative to that with rectangular light-to-dark transition on the daily profiles of Per1 and Per2 mRNA (in situ hybridization) and PER1 and PER2 protein (immunohistochemistry) levels within the rostral, middle and caudal regions of the mouse SCN. Under the short but not under the long photoperiod, Per1, Per2 and PER1, PER2 profiles were significantly phase-advanced under the twilight relative to rectangular light-to-dark transition in all SCN regions examined. Under the photoperiods with rectangular light-to-dark transition, Per1 and Per2 mRNA profiles in the caudal SCN were phase-advanced as compared with those in the rostral SCN. The phase differences between the SCN regions were reduced under the long, or completely abolished under the short, photoperiods with twilight. The data indicate that the twilight photoperiod provides stronger synchronization among the individual SCN cell subpopulations than the rectangular one, and the effect is more pronounced under the short than under the long photoperiod.
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Temporal gradient in the clock gene and cell-cycle checkpoint kinase Wee1 expression along the gut.
Chronobiol. Int.
PUBLISHED: 05-16-2009
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Circadian clocks were recently discovered in the rat and mouse colon as well as mouse stomach and jejunum. The aim of this study was to determine whether clocks in the upper part of the gut are synchronized with those in the lower part, or whether there is a difference in their circadian phases. Moreover, the profiles of core clock-gene expression were compared with the profiles of the clock-driven Wee1 gene expression in the upper and lower parts of the gut. Adult rats were transferred to constant darkness on the day of sampling. 24 h expression profiles of the clock genes Per1, Per2, Rev-erbalpha, and Bmal1 and the cell-cycle regulator Wee1 were examined by a reverse transcriptase-polymerase chain reaction within the epithelium of the rat duodenum, ileum, jejunum, and colon. In contrast to the duodenum, the rhythms in expression of all genes but Rev-erbalpha and Bmal1 in the colon exhibited non-sinusoidal profiles. Therefore, a detailed analysis of the gene expression every 1 h within the 12 h interval corresponding to the previous lights-on was performed. The data demonstrate that rhythmic profiles of the clock gene Per1, Per2, Bmal1, Rev-erbalpha, and clock-driven Wee1 expression within the epithelium from different parts of the rat gut exhibited a difference in phasing, such that the upper part of the gut, as represented by the duodenum, was phase-advanced to the lower part, as represented by the distal colon. Our data demonstrate that the circadian clocks within each part of the gut are mutually synchronized with a phase delay in the cranio-caudal axis. Moreover, they support the view that the individual circadian clocks may control the timing of cell cycle within different regions of the gut.
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Development of the light sensitivity of the clock genes Period1 and Period2, and immediate-early gene c-fos within the rat suprachiasmatic nucleus.
Eur. J. Neurosci.
PUBLISHED: 02-19-2009
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The molecular mechanism underlying circadian rhythmicity within the suprachiasmatic nuclei (SCN) of the hypothalamus has two light-sensitive components, namely the clock genes Per1 and Per2. Besides, light induces the immediate-early gene c-fos. In adult rats, expression of all three genes is induced by light administered during the subjective night but not subjective day. The aim of the present study was to ascertain when and where within the SCN the photic sensitivity of Per1, Per2 and c-fos develops during early postnatal ontogenesis. The specific aim was to find out when the circadian clock starts to gate photic sensitivity. The effect of a light pulse administered during either the subjective day or the first or second part of the subjective night on gene expression within the rat SCN was determined at postnatal days (P) 1, 3, 5 and 10. Per1, Per2 and c-fos mRNA levels were assessed 30 min, 1 and 2 h after the start of each light pulse by in situ hybridization histochemistry. Expression of Per1 and c-fos was light responsive from P1, and the responses began to be gated by the circadian clock at P3 and P10, respectively. Expression of Per2 was only slightly light responsive at P3, and the response was not fully gated until P5. These data demonstrate that the light sensitivity of the circadian clock develops gradually during postnatal ontogenesis before the circadian clock starts to control the response. The photoinduction of the clock gene Per2 develops later than that of Per1.
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Early chronotype and tissue-specific alterations of circadian clock function in spontaneously hypertensive rats.
PLoS ONE
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Malfunction of the circadian timing system may result in cardiovascular and metabolic diseases, and conversely, these diseases can impair the circadian system. The aim of this study was to reveal whether the functional state of the circadian system of spontaneously hypertensive rats (SHR) differs from that of control Wistar rat. This study is the first to analyze the function of the circadian system of SHR in its complexity, i.e., of the central clock in the suprachiasmatic nuclei (SCN) as well as of the peripheral clocks. The functional properties of the SCN clock were estimated by behavioral output rhythm in locomotor activity and daily profiles of clock gene expression in the SCN determined by in situ hybridization. The function of the peripheral clocks was assessed by daily profiles of clock gene expression in the liver and colon by RT-PCR and in vitro using real time recording of Bmal1-dLuc reporter. The potential impact of the SHR phenotype on circadian control of the metabolic pathways was estimated by daily profiles of metabolism-relevant gene expression in the liver and colon. The results revealed that SHR exhibited an early chronotype, because the central SCN clock was phase advanced relative to light/dark cycle and the SCN driven output rhythm ran faster compared to Wistar rats. Moreover, the output rhythm was dampened. The SHR peripheral clock reacted to the dampened SCN output with tissue-specific consequences. In the colon of SHR the clock function was severely altered, whereas the differences are only marginal in the liver. These changes may likely result in a mutual desynchrony of circadian oscillators within the circadian system of SHR, thereby potentially contributing to metabolic pathology of the strain. The SHR may thus serve as a valuable model of human circadian disorders originating in poor synchrony of the circadian system with external light/dark regime.
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Circadian system from conception till adulthood.
Prog. Brain Res.
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In mammals, the circadian system is composed of the central clock in the hypothalamic suprachiasmatic nuclei and of peripheral clocks that are located in other neural structures and in cells of the peripheral tissues and organs. In adults, the system is hierarchically organized so that the central clock provides the other clocks in the body with information about the time of day. This information is needed for the adaptation of their functions to cyclically changing external conditions. During ontogenesis, the system undergoes substantial development and its sensitivity to external signals changes. Perinatally, maternal cues are responsible for setting the phase of the developing clock, while later postnatally, the LD cycle is dominant. The central clock attains its functional properties during a gradual and programmed process. Peripheral clocks begin to exhibit rhythmicity independent of each other at various developmental stages. During the early developmental stages, the peripheral clocks are set or driven by maternal feeding, but later the central clock becomes fully functional and begins to entrain the periphery. During the perinatal period, the central and peripheral clocks seem to be vulnerable to disturbances in external conditions. Further studies are needed to understand the processes of how the circadian system develops and what degree of plasticity and resilience it possesses during ontogenesis. These data may lead to an assessment of the contribution of disturbances of the circadian system during early ontogenesis to the occurrence of circadian diseases in adulthood.
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An association between clock genes and clock-controlled cell cycle genes in murine colorectal tumors.
Int. J. Cancer
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Disruption of circadian machinery appears to be associated with the acceleration of tumor development. To evaluate the function of the circadian clock during neoplastic transformation, the daily profiles of the core clock genes Per1, Per2, Rev-Erb? and Bmal1, the clock-controlled gene Dbp and the clock-controlled cell cycle genes Wee1, c-Myc and p21 were detected by real-time RT-PCR in chemically induced primary colorectal tumors, the surrounding normal tissue and in the liver. The circadian rhythmicity of Per1, Per2, Rev-Erb? and Dbp was significantly reduced in tumor compared with healthy colon and the rhythmicity of Bmal1 was completely abolished. Interestingly, the circadian expression of Per1, Per2, Rev-Erb? and Dbp persisted in the colonic tissue surrounding the tumor but the rhythmic expression of Bmal1 was also abolished. Daily profiles of Wee1, c-Myc and p21 did not exhibit any rhythmicity either in tumors or in the colon of healthy animals. The absence of diurnal rhythmicity of cell cycle genes was partially associated with ageing, because young healthy mice showed rhythmicity in the core clock genes as well as in the Wee1 and p21. In the liver of tumor-bearing mice the clock gene rhythms were temporally shifted. The data suggest that the circadian regulation is distorted in colonic neoplastic tissue and that the gene-specific disruption may be also observed in the non-neoplastic tissues. These findings reinforce the role of peripheral circadian clockwork disruption for carcinogenesis and tumor progression.
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Photoperiodic modulation of the hepatic clock by the suprachiasmatic nucleus and feeding regime in mice.
Eur. J. Neurosci.
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Changes in photoperiod modulate the central circadian clock in the suprachiasmatic nucleus (SCN) as well as the peripheral clocks. Consequently, the SCN-driven output rhythms in activity and feeding are also modulated by the photoperiod. The aim of the present study was to elucidate whether photoperiodic modulation of the hepatic clock is mediated by changes in feeding or by another SCN-driven pathway. Five days after the change from short photoperiod (SP) to long photoperiod (LP), the profiles of Per2 and Rev-erb? expression in the rostral, middle and caudal regions of the SCN were desynchronized and those in the liver were modulated as in mice fully entrained to LP. The SCN profiles were not affected in mice left under SP and subjected to the 6-h night-time feeding regime for 5 days. In the liver, the profiles were shifted to the same phase, but their waveforms were not modulated compared with those under LP. In mice subjected to the change from SP to LP and fed twice daily during the daytime, the profiles in the SCN were not affected, whereas the waveforms and phases of those in the liver were affected. The data demonstrate that the adjustment of gene expression profiles in the rostral, middle and caudal SCN to the change from SP to LP proceeds within 5 days and is not affected by changes in the feeding regime. The results also suggest that the photoperiod-modulated SCN affects waveforms of gene expression profiles in the liver by food-independent signals.
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