Expression Of Circadian Rhythms Research Articles

Vasopressin: more than just an output of the circadian pacemaker? Focus on “Vasopressin receptor V1a regulates circadian rhythms of locomotor activity and expression of clock-controlled genes in the suprachiasmatic nuclei”

the timing of physiological events is carefully controlled so that incompatible processes do not compete and complementary functions coincide. Given the pivotal role of the hypothalamus in homeostatic regulation, the discovery that a master circadian pacemaker resides in this region was not

Vasopressin receptor V1a regulates circadian rhythms of locomotor activity and expression of clock-controlled genes in the suprachiasmatic nuclei

The suprachiasmatic nuclei (SCN) serve as the principal circadian pacemakers that coordinate daily cycles of behavior and physiology for mammals. A network of transcriptional and translational feedback loops underlies the operating molecular mechanism for circadian oscillation within the SCN neurons. It remains unclear how timing information is transmitted from SCN neurons to eventually evoke circadian rhythms. Intercellular communication between the SCN and its target neurons is critical for the generation of coherent circadian rhythms. At the molecular level, neuropeptides encoded by clock-controlled genes have been indicated as important output mediators. Arginine vasopressin (AVP) is the product of one such clock-controlled gene. Previous studies have demonstrated a circadian rhythm of AVP levels in the cerebrospinal fluid and the SCN. The physiological effects of AVP are mediated by three types of AVP receptors, designated as V1a, V1b, and V2. In this study, we report that V1a mRNA levels displayed a circadian rhythm in the SCN, peaking during night hours. The circadian rhythmicity of locomotor activities was significantly reduced in V1a-deficient (V1a(-/-)) mice (50-75% reduction in the power of fast Fourier transformation). However, the light masking and light-induced phase shift effects are intact in V1a(-/-) mice. Whereas the expression of clock core genes was unaltered, the circadian amplitude of prokineticin 2 (PK2) mRNA oscillation was attenuated in the SCN of V1a(-/-) mice ( approximately 50% reduction in the peak levels). In vitro experiments demonstrated that AVP, acting through V1a receptor, was able to enhance the transcriptional activity of the PK2 promoter. These studies thus indicate that AVP-V1a signaling plays an important role in the generation of overt circadian rhythms.

Energy-responsive timekeeping

An essential component of energy homeostasis lies in an organism's ability to coordinate daily patterns in activity, feeding, energy utilization and energy storage across the daily 24-h cycle. Most tissues of the body contain the molecular clock machinery required for circadian oscillation and rhythmic gene expression. Under normal circumstances, behavioural and physiological rhythms are orchestrated and synchronized by the suprachiasmatic nucleus (SCN) of the hypothalamus, considered to be the master circadian clock. However, metabolic processes are easily decoupled from the primarily light-driven SCN when food intake is desynchronized from normal diurnal patterns of activity. This dissociation from SCN based timing demonstrates that the circadian system is responsive to changes in energy supply and metabolic status. There has long been evidence for the existence of an anatomically distinct and autonomous food-entrainable oscillator (FEO) that can govern behavioural rhythms, when feeding becomes the dominant entraining stimulus. But now rapidly growing evidence suggests that core circadian clock genes are involved in reciprocal transcriptional feedback with genetic regulators of metabolism, and are directly responsive to cellular energy supply. This close interaction is likely to be critical for normal circadian regulation of metabolism, and may also underlie the disruption of proper metabolic rhythms observed in metabolic disorders, such as obesity and type-II diabetes.

Endogenous melatonin provides an effective circadian message to both the suprachiasmatic nuclei and the pars tuberalis of the rat

The suprachiasmatic nuclei (SCN) distribute the circadian neural message to the pineal gland which transforms it into a humoral circadian message, the nocturnal melatonin synthesis, which in turn modulates tissues expressing melatonin receptors such as the SCN or the pars tuberalis (PT). Nuclear orphan receptors (NOR), including rorbeta and rev-erbalpha, have been presented as functional links between the positive and negative loops of the molecular clock. Recent findings suggest that these NOR could be the initial targets of melatonin's chronobiotic message within the SCN. We investigated the role of these NOR in the physiological effect of endogenous melatonin on these tissues. We monitored rorbeta and rev-erbalpha mRNA expression levels by quantitative in situ hybridization after pinealectomy. Pinealectomy had no effect on NOR circadian expression rhythms in the SCN in 8-day pinealectomized (PX) animals. However in animals PX for 3 months, significant desynchronization between per1 and per2 transcription patterns appeared. These results suggest that endogenous melatonin could sustain the circadian rhythmicity and the phase relationship between the molecular partners of the SCN circadian system on a long-term basis. On the other hand, pinealectomy decreased the level and abolished the rhythmicity of NOR mRNA expression in the PT. These effects were partially prevented by daily melatonin administration in the drinking water. These results show that NOR can be regulated by the melatonin circadian rhythm in the PT and could be the link between the physiological action of melatonin and the core of the molecular circadian clock in this tissue.

CLOCK Is Required for Maintaining the Circadian Rhythms of Opsin mRNA Expression in Photoreceptor Cells

In zebrafish, the expression of long-wavelength cone (LC) opsin mRNA fluctuated rhythmically between the day and night. In a 24-h period, expression was high in the afternoon and low in the early morning. This pattern of fluctuation persisted in zebrafish that were kept in constant darkness, suggesting an involvement of circadian clocks. Functional expression of Clock, a circadian clock gene that contributes to the central circadian pacemaker, was found to play an important role in maintaining the circadian rhythms of LC opsin mRNA expression. In zebrafish embryos, in which the translation of Clock was inhibited by anti-Clock morpholinos, the circadian rhythms of LC opsin mRNA expression diminished. CLOCK may regulate the circadian rhythms of LC opsin mRNA expression via cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. In control retinas, the concentration of cAMP was high in the early morning and low in the remainder of the day and night. Inhibition of Clock translation abolished the fluctuation in the concentration of cAMP, thereby diminishing the circadian rhythms of opsin mRNA expression. Transient increase of cAMP concentrations in the early morning (i.e. by treating the embryos with 8-bromo-cAMP) restored the circadian rhythms of LC opsin mRNA expression in morpholino-treated embryos. Together, the data suggest that Clock plays important roles in regulating the circadian rhythms in photoreceptor cells.

Circadian Expression Profiles of Drug-Processing Genes and Transcription Factors in Mouse Liver

Temporal coordination of hepatic drug-processing gene (DPG) expression facilitates absorption, biotransformation, and excretion of exogenous and endogenous compounds. To further elucidate the circadian rhythm of hepatic DPG expression, male C57BL/6 mice were subjected to a standard 12-h light/dark cycle, and livers were collected at 2:00, 6:00, and 10:00 AM and 2:00, 6:00, and 10:00 PM. The mRNAs of hepatic phase I enzymes (cytochromes P450, aldehyde dehydrogenases, and carboxylesterases), phase II enzymes (glucuronosyltransferases, sulfotransferases, and glutathione S-transferases), uptake and efflux transporters, and transcription factors were quantified. Messenger RNAs of various genes were graphed across time of day and compared by hierarchical clustering. In general, the mRNA of phase I enzymes increased during the dark phase, whereas the mRNAs of most phase II enzymes and transporters reached maximal levels during the light phase. The majority of hepatic transcription factors exhibited expression peaks either before or after the onset of the dark phase. During the same time period, the negative clock regulator gene Rev-Erbalpha and the hepatic clock-controlled gene Dbp also reached mRNA expression peaks. Considering their important role in xenobiotic metabolism, hepatic transcription factors, such as constitutive androstane receptor, pregnane X receptor, aryl hydrocarbon receptor, and peroxisomal proliferator activated receptor alpha, may be involved in coupling the hepatic circadian clock to environmental cues. Taken together, these data demonstrate that the circadian expression of the DPG battery and transcription factors contribute to the temporal detoxification cycle in the liver.

Influence of chronotype and social zeitgebers on sleep/wake patterns

Inter-individual differences in the phase of the endogenous circadian rhythms have been established. Individuals with early circadian phase are called morning types; those with late circadian phase are evening types. The Horne and Ostberg Morningness-Eveningness Questionnaire (MEQ) is the most frequently used to assess individual chronotype. The distribution of MEQ scores is likely to be biased by several fact, ors, such as gender, age, genetic background, latitude, and social habits. The objective of the present study was to determine the effect of different social synchronizers on the sleep/wake cycle of persons with different chronotypes. Volunteers were selected from a total of 1232 UFPR undergraduate students who completed the MEQ. Thirty-two subjects completed the study, including 8 morning types, 8 evening types and 16 intermediate types. Sleep schedules were recorded by actigraphy for 1 week on two occasions: during the school term and during vacation. Sleep onset and offset times, sleep duration, and mid-sleep time for each chronotype group were compared by the Mann-Whitney U-test separately for school term and vacation. School term and vacation data were compared by the Wilcoxon matched-pair test. Morning types showed earlier sleep times and longer sleep duration compared with evening types (23:00 +/- 44 and 508.9 +/- 50.27 vs 01:08 +/- 61.95 and 456.44 +/- 59.08, for the weekdays during vacation). During vacation, the subjects showed later sleep times, except for the morning types, who did not exhibit differences for sleep onset times. The results support the idea that social schedules have an impact on the expression of circadian rhythmicity but this impact depends on the individual chronotype.

Scheduled exposures to a novel environment with a running‐wheel differentially accelerate re‐entrainment of mice peripheral clocks to new light–dark cycles

Effects of scheduled exposures to novel environment with a running-wheel were examined on re-entrainment to 8 h shifted light-dark (LD) cycles of mouse circadian rhythms in locomotor activity and clock gene, Per1, expression in the suprachiasmatic nucleus (SCN) and peripheral tissues. Per1 expression was monitored by a bioluminescence reporter introduced into mice. The animals were exposed to the novel environment for 3 h from the shifted dark onset for four cycles and released into constant darkness. In the phase-advance shift, the circadian rhythm in locomotor activity fully re-entrained in the exposed group, whereas it was in transients in the control. On the other hand, the circadian rhythm of Per1 expression in the SCN almost completely re-entrained in both the control and exposed groups. In the skeletal muscle and lung, the circadian rhythm fully re-entrained in the exposed group, whereas the rhythms in the control did not. In the phase-delay shift, the circadian rhythms in locomotor activity and Per1 expression almost completely re-entrained in both groups. These findings indicate that the scheduled exposures to novel environment with a running-wheel differentially accelerate the re-entrainment of the mouse peripheral clocks to 8 h phase-advanced LD cycles.

The Clock Protein CCA1 and the bZIP Transcription Factor HY5 Physically Interact to Regulate Gene Expression in Arabidopsis

The circadian clock regulates the expression of an array of Arabidopsis genes such as those encoding the LIGHT-HARVESTING CHLOROPHYLL A/B (Lhcb) proteins. We have previously studied the promoters of two of these Arabidopsis genes--Lhcb1*1 and Lhcb1*3--and identified a sequence that binds the clock protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). This sequence, designated CCA1-binding site (CBS), is necessary for phytochrome and circadian responsiveness of these genes. In close proximity to this sequence, there exists a G-box core element that has been shown to bind the bZIP transcription factor HY5 in other light-regulated plant promoters. In the present study, we examined the importance of the interaction of transcription factors binding the CBS and the G-box core element in the control of normal circadian rhythmic expression of Lhcb genes. Our results show that HY5 is able to specifically bind the G-box element in the Lhcb promoters and that CCA1 can alter the binding activity of HY5. We further show that CCA1 and HY5 can physically interact and that they can act synergistically on transcription in a yeast reporter gene assay. An absence of HY5 leads to a shorter period of Lhcb1*1 circadian expression but does not affect the circadian expression of CATALASE3 (CAT3), whose promoter lacks a G-box element. Our results suggest that interaction of the HY5 and CCA1 proteins on Lhcb promoters is necessary for normal circadian expression of the Lhcb genes.

Temporal profile of circadian clock gene expression in a transplanted suprachiasmatic nucleus and peripheral tissues

The mammalian hypothalamic suprachiasmatic nucleus (SCN) is the master oscillator that regulates the circadian rhythms of the peripheral oscillators. Previous studies have demonstrated that the transplantation of embryonic SCN tissues into SCN-lesioned arrhythmic mice restores the behavioral circadian rhythms of these animals. In our present study, we examined the clock gene expression profiles in a transplanted SCN and peripheral tissues, and also analysed the circadian rhythm of the locomotor activity in SCN-grafted mice. These experiments were undertaken to elucidate whether the transplanted SCN generates a dynamic circadian oscillation and maintains the phase relationships that can be detected in intact mice. The grafted SCN indeed showed dynamic circadian expression rhythms of clock genes such as mPeriod1 (mPer1) and mPeriod2 (mPer2). Furthermore, the phase differences between the expression rhythms of these genes in the grafted SCN and the locomotor activity rhythms of the transplanted animals were found to be very similar to those in intact animals. Moreover, in the liver, kidney and skeletal muscles of the transplanted animals, the phase angles between the circadian rhythm of the grafted SCN and that of the peripheral tissues were maintained as in intact animals. However, in the SCN-grafted animals, the amplitudes of the mPer1 and mPer2 rhythms were attenuated in the peripheral tissues. Our current findings therefore indicate that a transplanted SCN has the capacity to generate a dynamic intrinsic circadian oscillation, and can also lock the normal phase angles among the SCN, locomotor activity and peripheral oscillators in a similar manner as in intact control animals.

Multifactorial Regulation of Daily Rhythms in Expression of the Metabolically Responsive GeneSpot14in the Mouse Liver

Spot14 is a putative transcriptional regulator for genes involved in fatty acid synthesis. The Spot14 gene is activated in response to lipogenic stimuli such as dietary carbohydrate and is also under circadian regulation. The authors investigated factors responsible for daily oscillation of Spot14 expression. If mice were kept under a 12-h light/12-h dark cycle with ad libitum feeding, Spot14 mRNA levels in the liver reached a peak at an early dark period when mice, as nocturnal animals, start feeding. Under fasting, while Spot14 mRNA levels were generally decreased, the rhythmicity was still maintained, suggesting contribution of both nutritional elements and circadian clock factors on robust rhythmicity of Spot14 expression. Effects of circadian clock factors were confirmed by the observations that the circadian rhythm of Spot14 expression was seen also under the constant darkness and that the rhythmicity was lost in Clock mutant mice. When mice were housed in short-photoperiod (6-h light/18-h dark) and long-photoperiod (18-h light/6-h dark) cycles, rhythms of Spot14 mRNA levels were phase advanced and phase delayed, respectively, being concordant with the notion that Spot14 expression is under the control of the light-entrainable oscillator. As for nutritional mediators, in the liver of db/db mice exhibiting hyperinsulinemia-accompanied hyperglycemia, Spot14 mRNA levels were constantly high without apparent rhythmicity, consistent with previous observations for strong activation of the Spot14 gene by glucose and insulin. Restricted feeding during the 4-h mid-light period caused a phase advance of the Spot14 expression rhythm. On the other hand, restricted feeding during the 4-h mid-dark period led to damping of the rhythmicity, apparently resulting from the separation of phases between effects of the light/dark cycle and feeding on Spot14 expression. Thus, the daily rhythm of Spot14 expression in the liver is under the control of the light-entrainable oscillator, food-entrainable oscillator, and food-derived nutrients, in a separate or cooperative manner.

Time to Grow: Circadian Clock Controls Plant Hormone Signaling and Response

Unable to move from one place to another in response to changing conditions, plants survive by moderating their growth to make the most of their circumstances. Plants sprout new leaves and roots and grow toward or away from light and gravity, all of which requires the growth hormone auxin. Buried in historical scientific literature are hints that auxin might affect plants differently at different times of day. This makes sense from an adaptive standpoint: an internal clock allows plants to respond to stimuli such as sun, rain, and being eaten in the context of regular rhythms of the inescapable world around them. But how do they do it? The mechanisms tuning growth to internal rhythms, with implications for everything from growing crops to protecting biodiversity in the face of global climate change, have remained a mystery. Now, Michael Covington and Stacey Harmer have discovered clues to the roots of rhythmic growth. The researchers began by identifying genes in Arabidopsis, the botanical equivalent of the laboratory rat, showing a circadian (daily) rhythm in transcription as indicated by changes in messenger RNA abundance. A surprising number of these genes are involved in auxin signaling. But at what stage? Looking closely at the genes involved, the researchers determined that auxin and the circadian clock are intimately intertwined virtually every step of the way. Genes regulating the production of auxin are clock controlled. So are genes that make auxin carriers, that inactivate auxin, and that make the receptor molecules that sense auxin and subsequently alter the transcription of other genes. Many of the genes auxin induces also show a circadian rhythm. To explore in more detail where the circadian clock and auxin signaling pathways intersect, the researchers used a luciferase reporter assay—basically, a firefly gene inserted into the genome that produces a glow when it and associated genes are activated. Covington and Harmer began by determining whether auxin affects circadian rhythms. Using the luciferase reporter assay to determine when genes controlling circadian rhythm were activated, they found that in the short term, added auxin didn’t affect the phase of the rhythm, but at some concentrations it increased period length and reduced amplitude (that is, the magnitude of fluctuation in the apparent activity of reporter genes). Prolonged auxin treatment slightly lengthened the period and dramatically reduced the amplitude of the rhythm. To determine whether rhythms affect auxin’s effects on gene transcription, the researchers inserted the firefly gene into plants in a way that caused them to glow when an auxin promoter, eDR5, is activated. These “eDR5::LUC” plants showed a circadian rhythm in reporter gene expression, indicating that auxin signaling is indeed clock controlled. The circadian regulation of auxin responses suggests that many aspects of plant physiology not previously thought to be under circadian control may show time-of-day-specific sensitivity. (Image: Michael F. Covington and Kimberly J. Cope) Next, the researchers explored whether the circadian fluctuation of auxin levels is what causes the rhythm in the transcription of auxin-related genes by administering auxin to eDR5::LUC plants. The plants responded with a quick glow, but not with change in phase or amplitude of glowing over the long term. The researchers then repeated the same experiment using plants in which auxin transport mechanisms were disrupted, with similar results. Their conclusion: the rhythms in transcription of auxin-controlled genes are not due to rhythms in auxin synthesis or transport. Circadian clocks have been found to alter responses to certain stimuli, a phenomenon known as “gating.” To find out whether the sensitivity of plants to exogenous auxin is altered by their biological clock, the researchers administered auxin to eDR5::LUC seedlings under constant light conditions. When auxin was given during day, the plants showed little if any increase in glowing; when it was given at night, they showed a clear response that peaked just before dawn. This differential response affirmed that auxin signaling is indeed gated by the plant’s internal rhythms. To determine whether gating shows up in growth patterns as well as in transcription, the researchers observed the growth of the embryonic shoot under continuous light in both control and auxin-treated seedlings. Control plants showed a rhythm in elongation. Auxin treatment boosted growth when given during what would have been night, but not during what would have been day—the same rhythm shown previously in transcription activation. The researchers concluded that growth is gated by auxin, too. Buoyed by their findings, the researchers look forward to exploring the interplay of rhythms and auxin action with respect to various aspects of plant development, including photo- and gravitropism and the development of specialized structures. Such studies will provide additional valuable insights into how circadian rhythms help plants adapt to the world around them.

Vasopressin immunoreactivity, but not vasoactive intestinal polypeptide, correlates with expression of circadian rhythmicity in the suprachiasmatic nucleus of voles

In common voles (Microtus arvalis), natural variation in locomotor behavior can be exploited to study the mechanism of pacemaker control over circadian timing of behavior. Here we studied daily patterns in numbers of neuropeptide immunoreactive suprachiasmatic nucleus neurons in rhythmic, weakly rhythmic, and non-rhythmic voles. Circadian rhythmic voles showed circadian variation in numbers of vasoactive intestinal polypeptide and vasopressin immunoreactive suprachiasmatic nucleus neurons with a peak at zeitgeber time 0. In contrast, voles with weak or no circadian rhythmicity exhibited similar fluctuations for vasoactive intestinal polypeptide, but a continuous, non-rhythmic high profile for vasopressin. Vole suprachiasmatic nucleus neurons do not produce somatostatin or substance P. We conclude that the vasopressin system in the common vole suprachiasmatic nucleus acts as a principal correlate with expression of circadian behavior, in contrast to vasoactive intestinal polypeptide, somatostatin, and substance P. We also conclude that high levels of vasopressin immunoreactivity in the non-rhythmic vole suprachiasmatic nucleus is in line with previously demonstrated hampered release, probably resulting in vasopressin accumulation in the suprachiasmatic nucleus. Vasopressin could be a candidate in mediating output of the vole circadian clock, leading to circadian expression of locomotor behavior.

MicroRNA Modulation of Circadian-Clock Period and Entrainment

microRNAs (miRNAs) are a class of small, noncoding RNAs that regulate the stability or translation of mRNA transcripts. Although recent work has implicated miRNAs in development and in disease, the expression and function of miRNAs in the adult mammalian nervous system have not been extensively characterized. Here, we examine the role of two brain-specific miRNAs, miR-219 and miR-132, in modulating the circadian clock located in the suprachiasmatic nucleus. miR-219 is a target of the CLOCK and BMAL1 complex, exhibits robust circadian rhythms of expression, and the in vivo knockdown of miR-219 lengthens the circadian period. miR-132 is induced by photic entrainment cues via a MAPK/CREB-dependent mechanism, modulates clock-gene expression, and attenuates the entraining effects of light. Collectively, these data reveal miRNAs as clock- and light-regulated genes and provide a mechanistic examination of their roles as effectors of pacemaker activity and entrainment.

Circadian Rhythms: Per2bations in the Liver Clock

A master circadian clock resides in the brain and is required to synchronize the clocks in peripheral tissues such as the liver. Until now, it has been unclear how the central clock synchronizes the peripheral ones. New work points to one of the core clock genes, mPer2, as an essential link in this chain.

PER3 Polymorphism Predicts Sleep Structure and Waking Performance

Circadian rhythmicity and sleep homeostasis interact to regulate sleep-wake cycles [1-4], but the genetic basis of individual differences in sleep-wake regulation remains largely unknown [5]. PERIOD genes are thought to contribute to individual differences in sleep timing by affecting circadian rhythmicity [6], but not sleep homeostasis [7, 8]. We quantified the contribution of a variable-number tandem-repeat polymorphism in the coding region of the circadian clock gene PERIOD3 (PER3) [9, 10] to sleep-wake regulation in a prospective study, in which 24 healthy participants were selected only on the basis of their PER3 genotype. Homozygosity for the longer allele (PER3(5/5)) had a considerable effect on sleep structure, including several markers of sleep homeostasis: slow-wave sleep (SWS) and electroencephalogram (EEG) slow-wave activity in non-rapid eye movement (non-REM) sleep and theta and alpha activity during wakefulness and REM sleep were all increased in PER3(5/5) compared to PER3(4/4) individuals. In addition, the decrement of cognitive performance in response to sleep loss was significantly greater in the PER3(5/5) individuals. By contrast, the circadian rhythms of melatonin, cortisol, and peripheral PER3 mRNA expression were not affected. The data show that this polymorphism in PER3 predicts individual differences in the sleep-loss-induced decrement in performance and that this differential susceptibility may be mediated by its effects on sleep homeostasis.

Hippocampal 11β-Hydroxysteroid Dehydrogenase Type 1 Messenger Ribonucleic Acid Expression Has a Diurnal Variability that Is Lost in the Obese Zucker Rat

Circulating levels of glucocorticoids show a circadian rhythm. Obesity is associated with a flattening of the diurnal rhythm; plasma cortisol levels are slightly increased during the trough, although they are normal or low in the morning. Studies in humans and in leptin-resistant Zucker rats suggest that tissue-specific alterations in glucocorticoid exposure might play a key role for development of obesity and obesity-associated dysregulation of the hypothalamic-pituitary-adrenal axis. We hypothesized that there is a circadian rhythm in prereceptor metabolism of glucocorticoids exerted by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in brain and/or peripheral tissues (liver, fat, and muscle) that might be abrogated in obesity. The present study demonstrates a circadian rhythm in 11beta-HSD1 mRNA expression (35-45% increase at morning vs. evening, P < 0.05) in dentate gyrus granular layer and CA1 subregions of the hippocampus in lean Zucker rats that was lost in the obese rats. Sprague Dawley rats also revealed a diurnal rhythm in hippocampal 11beta-HSD1 mRNA expression. There was no circadian variation in 11beta-HSD enzyme activity in peripheral tissues, although obese Zucker rats had a decreased enzyme activity in liver and epididymal fat (by approximately 40%, P < 0.05) compared with lean rats. In Sprague Dawley rats, 11beta-HSD activity in adipose tissue was higher in retroperitoneal and epididymal vs. sc fat (P < 0.001). In summary, obese Zucker rats lack a circadian rhythm of 11beta-HSD1 gene expression in the hippocampus, which may contribute to increased activity of the hypothalamic-pituitary-adrenal axis and altered diurnal variation of circulating corticosterone levels.

Involvement of Indoxyl Sulfate in Renal and Central Nervous System Toxicities During Cisplatin-induced Acute Renal Failure

The purpose of the present study was to explore the involvement of indoxyl sulfate (IS) in nephrotoxicity and central nervous system (CNS) toxicity in cisplatin (CDDP)-treated rats. Renal function was evaluated by serum creatinine and BUN levels. The IS levels in the serum, brain and kidney was monitored by high-performance liquid chromatography method. Body weight and rectal temperature were monitored. Real-time PCR analysis was performed to examine rPer2 mRNA expression. Renal function deteriorated in a time-dependent manner after administration of CDDP. The concentration of IS in the serum, brain and kidney markedly increased 24-84 h after commencement of CDDP treatment. The observed increase in the levels of serum creatinine, BUN and IS was suppressed by concomitant administration of AST-120. Rectal temperature was significantly lowered 72-92 h after CDDP-treatment, which was partially restored by coadministration of AST-120. Moreover, the amplitude of rectal temperature rhythms was disrupted by treatment with CDDP. Circadian rhythm of rPer2 mRNA expression, a clock gene, in suprachiasmatic nucleus (SCN) and kidney was disturbed in CDDP-treated rats. An increase in the IS level and the associated disturbance to the circadian rhythm are involved in the renal and CNS toxicities in CDDP-treatment.

Synchronizing multiphasic circadian rhythms of rhodopsin promoter expression in rod photoreceptor cells

Endogenous circadian clocks regulate day-night rhythms of animal behavior and physiology. In zebrafish, the circadian clocks are located in the pineal gland and the retina. In the retina, each photoreceptor is considered a circadian oscillator. A critical question is whether the individual circadian oscillators are synchronized. If so, the mechanism that underlies the synchronization needs to be elucidated. We generated a transgenic zebrafish line that expresses short half-life GFP under the transcriptional control of the rhodopsin promoter. Time-lapse imaging of rhodopsin promoter-driven GFP expression revealed that during 24 h in constant darkness, rhodopsin promoter expression in rod photoreceptor cells fluctuated rhythmically. However, the pattern of fluctuation differed between individual cells. In some cells, peak expression was seen in the subjective early morning, whereas in other cells, peak expression was seen in the afternoon or at night. Light transiently decreased rhodopsin expression, thereby synchronizing the multiphasic circadian oscillation. The application of dopamine or dopamine D2 receptor agonist also synchronized the circadian rhythms of rhodopsin promoter expression. When the D2 receptors were pharmacologically blocked, light exposure produced no effect. This suggests that the synchronization of the circadian rhythms of rhodopsin promoter expression by light is mediated by dopamine D2 receptors. The mechanism that underlies the synchronization probably involves dopamine-mediated Ca2+ signaling pathways. Light, as well as dopamine, lowered Ca2+ influx into the rod cells, thereby resetting rhodopsin promoter expression to the initial phase.

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.