Modification of the Per2 clock gene in mPer2Luc reporter mice significantly alters circadian function. Behavioral period in constant dark is lengthened, and dissociates into two distinct components in constant light. Rhythms exhibit increased bimodality, enhanced phase resetting to light pulses, and altered entrainment to scheduled feeding. Mechanistic mathematical modelling predicts that enhanced protein interactions with the modified mPER2 C-terminus, combined with differential clock regulation among SCN subregions, can account for effects on circadian behavior via increased Per2 transcript and protein stability. PER2::LUC produces greater suppression of CLOCK:BMAL1 E-box activity than PER2. mPer2Luc carries a 72 bp deletion in exon 23 of Per2, and retains a neomycin resistance cassette that affects rhythm amplitude but not period. The results show that mPer2Luc acts as a circadian clock mutation illustrating a need for detailed assessment of potential impacts of c-terminal tags in genetically modified animal models.

• MeSH
• chování zvířat MeSH
• cirkadiánní proteiny Period genetika   MeSH
• cirkadiánní rytmus * MeSH
• lokomoce MeSH
• luciferasy genetika   MeSH
• mutace MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• stravovací zvyklosti MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Light entrains the master circadian clock in the suprachiasmatic nucleus (SCN) predominantly through glutamatergic signaling via NMDA receptors. The magnitude and the direction of resulting phase shifts depend on timing of the photic stimulus. Previous reports based on behavioral and electrophysiological data suggested that endocannabinoids (EC) might reduce the ability of the SCN clock to respond to light. However, there is little direct evidence for the involvement of EC in entrainment of the rhythmic clock gene expression in the SCN. We have used luminescence recording of cultured SCN slices from mPer2Luc mice to construct a complete phase response curve (PRC) for NMDA receptor activation. The results demonstrated that NMDA administration phase-shifts the PER2 rhythm in a time-specific manner. A stable "singularity," in the course of which the clock seemingly stops while the overall phase is caught between delays and advances, can occur in response to NMDA at a narrow interval during the PER2 level decrease. NMDA-induced phase delays were affected neither by the agonist (WIN 55,212-2 mesylate) nor by the antagonist (rimonabant hydrochloride) of EC receptors. However, the agonist significantly reduced the NMDA-induced phase advance of the clock, while the antagonist enhanced the phase advance, causing a shift in the sensitivity window of the SCN to NMDA. The modulation of EC signaling in the SCN had no effect by itself on the phase of the PER2 rhythm. The results provide evidence for a modulatory role of EC in photic entrainment of the circadian clock in the SCN.

• Publikační typ
• časopisecké články MeSH

The physiological function of the pancreas is controlled by the circadian clock. The aim of this study was to determine whether aging-induced changes in glucose homeostasis affect properties of the circadian clock in the pancreas and/or its sensitivity to disturbances in environmental lighting conditions. mPer2Luc mice aged 24-26 months developed hyperinsulinemic hypoglycaemia, which was likely due to the Pclo-mediated insulin hyper-secretion and Slc2a2-mediated glucose transport impairment in the pancreas, and due to the alterations in Pp1r3c-related glycogen storage and Sgk1-related glucose transport in the liver. In the pancreatic tissue, aging affected clock gene expression only marginally, it upregulated Bmal1 and downregulated Clock expression. Whereas aging significantly impaired the circadian clock in lung explants, which were used as a control tissue, the properties of the pancreatic clock in vitro were not affected. The data suggest a non-circadian role of Bmal1 in changes of pancreatic function that occur during aging. Additionally, the pancreatic clock was more sensitive to exposure of animals to constant light conditions. These findings provide an explanation for the previously demonstrated relationship between disturbances in the circadian system and disordered glucose homeostasis, including diabetes mellitus type 2, in subjects exposed to long-term shift work.

• MeSH
• cirkadiánní hodiny * účinky záření   MeSH
• cirkadiánní proteiny Period metabolismus   MeSH
• glukosa metabolismus   MeSH
• homeostáza * MeSH
• játra metabolismus   MeSH
• kolon metabolismus   MeSH
• myši MeSH
• orgánová specificita genetika   MeSH
• pankreas metabolismus   účinky záření   MeSH
• proteiny CLOCK genetika   metabolismus   MeSH
• regulace genové exprese účinky záření   MeSH
• stárnutí metabolismus   MeSH
• světlo MeSH
• transkripční faktory ARNTL genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

INTRODUCTION: The maternal part of the rodent placenta harbors a circadian clock which robustly responds to glucocorticoids, however, its sensitivity to other hormones has not been elucidated. In this study, we tested five selected hormones (dopamine, melatonin, insulin, leptin and ghrelin) for their effectiveness to affect the clock in decidual region of mouse placenta in vitro. METHODS: We administered the hormones or corresponding vehicles at various time points over 24 h to organotypic placental explants of mPer2Luc mice containing the decidua basalis (DB) region and monitored their effects on amplitude, period, median expression level (mesor) and phase of PER2-driven bioluminescence rhythms. RESULTS: Dopamine significantly increased the amplitude, robustly dampened the mesor, and during a narrow time interval (corresponding to daytime) induced phase delays of the rhythms. In contrast, melatonin had no effect on amplitude, but induced phase advances of the rhythms at the opposite time window than dopamine (corresponding to nighttime). Leptin and ghrelin, but not insulin, slightly increased amplitudes and moderately modulated phase delays of the clock, suggesting that the DB clock, in contrast to other peripheral clocks, is rather resilient to abrupt changes in levels of feeding- and metabolism-related hormones. DISCUSSION: The results demonstrate for the first time that dopamine and melatonin exhibit delicate yet specific effects on parameters of the DB clock and may thus potentially contribute to fine-tuning of its phase under in vivo conditions. It also implies that dysregulation of their levels, which accompany various pathologies, may account for malfunction of the clock in DB.

• MeSH
• cirkadiánní hodiny * MeSH
• cirkadiánní rytmus * MeSH
• dopamin fyziologie   MeSH
• hormony fyziologie   MeSH
• myši MeSH
• placenta metabolismus   MeSH
• těhotenství MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Choroid plexus (ChP), the brain structure primarily responsible for cerebrospinal fluid production, contains a robust circadian clock, whose role remains to be elucidated. The aim of our study was to [1] identify rhythmically controlled cellular processes in the mouse ChP and [2] assess the role and nature of signals derived from the master clock in the suprachiasmatic nuclei (SCN) that control ChP rhythms. To accomplish this goal, we used various mouse models (WT, mPer2Luc, ChP-specific Bmal1 knockout) and combined multiple experimental approaches, including surgical lesion of the SCN (SCNx), time-resolved transcriptomics, and single cell luminescence microscopy. In ChP of control (Ctrl) mice collected every 4 h over 2 circadian cycles in darkness, we found that the ChP clock regulates many processes, including the cerebrospinal fluid circadian secretome, precisely times endoplasmic reticulum stress response, and controls genes involved in neurodegenerative diseases (Alzheimer's disease, Huntington's disease, and frontotemporal dementia). In ChP of SCNx mice, the rhythmicity detected in vivo and ex vivo was severely dampened to a comparable extent as in mice with ChP-specific Bmal1 knockout, and the dampened cellular rhythms were restored by daily injections of dexamethasone in mice. Our data demonstrate that the ChP clock controls tissue-specific gene expression and is strongly dependent on the presence of a functional connection with the SCN. The results may contribute to the search for a novel link between ChP clock disruption and impaired brain health.

• MeSH
• cirkadiánní hodiny * fyziologie   MeSH
• cirkadiánní rytmus fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• nucleus suprachiasmaticus * metabolismus   fyziologie   MeSH
• plexus chorioideus * metabolismus   fyziologie   MeSH
• transkripční faktory ARNTL metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Lithium is an effective mood stabilizer, but the mechanism of its therapeutic action is not well understood. We investigated the effect of lithium on the circadian clock located in the ventricle barrier complex containing the choroid plexus (CP), a part of the glymphatic system that influences gross brain function via the production of cerebrospinal fluid. The mPer2Luc mice were injected with lithium chloride (LiCl) or vehicle, and their effects on the clock gene Nr1d1 in CP were detected by RT qPCR. CP organotypic explants were prepared to monitor bioluminescence rhythms in real time and examine the responses of the CP clock to LiCl and inhibitors of glycogen synthase kinase-3 (CHIR-99021) and protein kinase C (chelerythrine). LiCl affected Nr1d1 expression levels in CP in vivo and dose-dependently delayed the phase and prolonged the period of the CP clock in vitro. LiCl and CHIR-99021 had different effects on 1] CP clock parameters (amplitude, period, phase), 2] dexamethasone-induced phase shifts of the CP clock, and 3] dynamics of PER2 degradation and de novo accumulation. LiCl-induced phase delays were significantly reduced by chelerythrine, suggesting the involvement of PKC activity. The effects on the CP clock may be involved in the therapeutic effects of lithium and hypothetically improve brain function in psychiatric patients by aligning the function of the CP clock-related glymphatic system with the sleep-wake cycle. Importantly, our data argue for personalized timing of lithium treatment in BD patients.

• MeSH
• cirkadiánní hodiny * MeSH
• cirkadiánní proteiny Period genetika   MeSH
• cirkadiánní rytmus genetika   MeSH
• lithium farmakologie   MeSH
• myši MeSH
• plexus chorioideus metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

AIMS: Circadian clocks in the hippocampus (HPC) align memory processing with appropriate time of day. Our study was aimed at ascertaining the specificity of glycogen synthase kinase 3-beta (GSK3β)- and glucocorticoid (GC)-dependent pathways in the entrainment of clocks in individual HPC regions, CA1-3, and dentate gyrus (DG). METHODS: The role of GCs was addressed in vivo by comparing the effects of adrenalectomy (ADX) and subsequent dexamethasone (DEX) supplementation on clock gene expression profiles (Per1, Per2, Nr1d1, and Bmal1). In vitro the effects of DEX and the GSK3β inhibitor, CHIR-99021, were assessed from recordings of bioluminescence rhythms in HPC organotypic explants of mPER2Luc mice. RESULTS: Circadian rhythms of clock gene expression in all HPC regions were abolished by ADX, and DEX injections to the rats rescued those rhythms in DG. The DEX treatment of the HPC explants significantly lengthened periods of the bioluminescence rhythms in all HPC regions with the most significant effect in DG. In contrast to DEX, CHIR-99021 significantly shortened the period of bioluminescence rhythm. Again, the effect was most significant in DG which lacks the endogenously inactivated (phosphorylated) form of GSK3β. Co-treatment of the explants with CHIR-99021 and DEX produced the CHIR-99021 response. Therefore, the GSK3β-mediated pathway had dominant effect on the clocks. CONCLUSION: GSK3β- and GC-dependent pathways entrain the clock in individual HPC regions by modulating their periods in an opposite manner. The results provide novel insights into the mechanisms connecting the arousal state-relevant signals with temporal control of HPC-dependent memory and cognitive functions.

• MeSH
• cirkadiánní hodiny * genetika   MeSH
• cirkadiánní proteiny Period genetika   metabolismus   MeSH
• cirkadiánní rytmus MeSH
• glukokortikoidy metabolismus   farmakologie   MeSH
• gyrus dentatus metabolismus   MeSH
• hipokampus metabolismus   MeSH
• kinasa 3 glykogensynthasy metabolismus   farmakologie   MeSH
• kinasa glykogensynthasy 3beta metabolismus   MeSH
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The choroid plexus (ChP) in the brain ventricles has a major influence on brain homeostasis. In this study, we aimed to determine whether the circadian clock located in ChP is affected by chronodisruption caused by misalignment with the external light/dark cycle and/or inflammation. Adult mPer2Luc mice were maintained in the LD12:12 cycle or exposed to one of two models of chronic chronodisruption - constant light for 22-25 weeks (cLL) or 6-hour phase advances of the LD12:12 cycle repeated weekly for 12 weeks (cLD-shifts). Locomotor activity was monitored before the 4th ventricle ChP and suprachiasmatic nuclei (SCN) explants were recorded in real time for PER2-driven population and single-cell bioluminescence rhythms. In addition, plasma immune marker concentrations and gene expression in ChP, prefrontal cortex, hippocampus and cerebellum were analyzed. cLL dampened the SCN clock but did not shorten the inactivity interval (sleep). cLD-shifts had no effect on the SCN clock, but transiently affected sleep duration and fragmentation. Both chronodisruption protocols dampened the ChP clock. Although immune markers were elevated in plasma and hippocampus, levels in ChP were unaffected, and unlike the liver clock, the ChP clock was resistant to lipopolysaccharide treatment. Importantly, both chronodisruption protocols reduced glucocorticoid signaling in ChP. The data demonstrate the high resistance of the ChP clock to inflammation, highlighting its role in protecting the brain from neuroinflammation, and on the other hand its high sensitivity to chronodisruption. Our results provide a novel link between human lifestyle-induced chronodisruption and the impairment of ChP-dependent brain homeostasis.

• MeSH
• chronická lymfatická leukemie * MeSH
• cirkadiánní hodiny * MeSH
• cirkadiánní proteiny Period genetika   metabolismus   MeSH
• cirkadiánní rytmus fyziologie   MeSH
• lidé MeSH
• myši MeSH
• plexus chorioideus metabolismus   MeSH
• zánět MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH