N6-methyladenosine (m6A) is the most abundant epitranscriptomic mark that regulates the fate of RNA molecules. Recent studies have revealed a bidirectional interaction between m6A modification and the circadian clock. However, the precise temporal dynamics of m6A global enrichment in the central circadian pacemaker have not been fully elucidated. Our study investigates the relationship between FTO demethylase and molecular clocks in primary cells of the suprachiasmatic nucleus (SCN). In addition, we examined the effects of lipopolysaccharide (LPS) on Fto expression and the role of FTO in LPS-induced reactive oxygen species (ROS) production in primary SCN cell culture. We observed circadian rhythmicity in the global m6A levels, which mirrored the rhythmic expression of the Fto demethylase. Silencing FTO using siRNA reduced the mesor of Per2 rhythmicity in SCN primary cells and extended the period of the PER2 rhythm in SCN primary cell cultures from PER2::LUC mice. When examining the immune response, we discovered that exposure to LPS upregulated global m6A levels while downregulating Fto expression in SCN primary cell cultures. Interestingly, we found a loss of circadian rhythmicity in Fto expression following LPS treatment, indicating that the decrease of FTO levels may contribute to m6A upregulation without directly regulating its circadian rhythm. To explore potential protective mechanisms against neurotoxic inflammation, we examined ROS production following LPS treatment in SCN primary cell cultures pretreated with FTO siRNA. We observed a time-dependent pattern of ROS induction, with significant peak at 32 h but not at 20 h after synchronization. Silencing the FTO demethylase abolished ROS induction following LPS exposure, supporting the hypothesis that FTO downregulation serves as a protective mechanism during LPS-induced neuroinflammation in SCN primary cell cultures.

• MeSH
• adenosin * analogy a deriváty   metabolismus   MeSH
• cirkadiánní hodiny * účinky léků   fyziologie   genetika   MeSH
• cirkadiánní proteiny Period metabolismus   genetika   MeSH
• cirkadiánní rytmus účinky léků   fyziologie   MeSH
• gen pro FTO * metabolismus   genetika   MeSH
• kultivované buňky MeSH
• lipopolysacharidy * farmakologie   MeSH
• methylace RNA MeSH
• metylace účinky léků   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• neurozánětlivé nemoci metabolismus   MeSH
• nucleus suprachiasmaticus * metabolismus   účinky léků   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• RNA genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Suprachiasmatic nucleus (SCN) of the hypothalamus is the master clock that drives circadian rhythms in physiology and behavior and adjusts their timing to external cues. Neurotransmitter glutamate and glutamatergic receptors sensitive to N-methyl-d-aspartate (NMDA) play a dual role in the SCN by coupling astrocytic and neuronal single cell oscillators and by resetting their phase in response to light. Recent reports suggested that signaling by endogenous cannabinoids (ECs) participates in both of these functions. We have previously shown that ECs, such as 2-arachidonoylglycerol (2-AG), act via CB1 receptors to affect the SCN response to light-mimicking NMDA stimulus in a time-dependent manner. We hypothesized that this ability is linked to the circadian regulation of EC signaling. We demonstrate that circadian clock in the rat SCN regulates expression of 2-AG transport, synthesis and degradation enzymes as well as its receptors. Inhibition of the major 2-AG synthesis enzyme, diacylglycerol lipase, enhanced the phase delay and lowered the amplitude of explanted SCN rhythm in response to NMDAR activation. Using microscopic PER2 bioluminescence imaging, we visualized how individual single cell oscillators in different parts of the SCN respond to the DAGL inhibition/NMDAR activation and shape response of the whole pacemaker. Additionally, we present strong evidence that the zero amplitude behavior of the SCN in response to single NMDA stimulus in the middle of subjective night is the result of a loss of rhythm in individual SCN cells. The paper provides new insights into the modulatory role of endocannabinoid signaling during the light entrainment of the SCN.

• MeSH
• agonisté excitačních aminokyselin farmakologie   MeSH
• cirkadiánní rytmus účinky léků   fyziologie   MeSH
• endokanabinoidy fyziologie   MeSH
• krysa rodu rattus MeSH
• lipoproteinlipasa antagonisté a inhibitory   metabolismus   MeSH
• myši transgenní MeSH
• myši MeSH
• N-methylaspartát farmakologie   MeSH
• nucleus suprachiasmaticus cytologie   účinky léků   fyziologie   MeSH
• potkani Wistar MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The mammalian circadian pacemaker in the suprachiasmatic nucleus (SCN) regulates behavioral and physiological processes in a 24-h cycle. During its development, the SCN can be sensitive to external stimuli which may change the circadian phenotypes in adulthood. Here, we investigated the effects of prenatal exposure to endotoxin lipopolysaccharide (LPS) on the developing rhythms in expression of Per1, Per2, Nr1d1 and Rasd1 along the rostrocaudal axis of the SCN, and on the rhythm of the rate-limiting enzyme in melatonin synthesis, pineal alkylamine N-acetyltransferase (AA-NAT). The prenatal LPS treatment induced anxiety-like behavior in adulthood as shown before and affected the rhythmicity of clock genes in the SCN. The major effect was observed for Nr1d1 expression; the least affected gene was Per2. The Nr1d1 in the LPS-treated group was arrhythmic at postnatal day 3, but showed significantly higher amplitude at postnatal day 20 at all SCN parts, similarly to the AA-NAT activity in pineal glands, thus suggesting adaptive flexibility of the developing SCN to immune challenges in early development.

• MeSH
• arylalkylamin-N-acetyltransferasa metabolismus   MeSH
• chování zvířat účinky léků   MeSH
• cirkadiánní hodiny účinky léků   MeSH
• cirkadiánní proteiny Period účinky léků   metabolismus   MeSH
• epifýza mozková účinky léků   metabolismus   MeSH
• krysa rodu rattus MeSH
• lipopolysacharidy toxicita   MeSH
• nucleus suprachiasmaticus účinky léků   MeSH
• potkani Wistar MeSH
• těhotenství MeSH
• úzkost MeSH
• zpožděný efekt prenatální expozice chemicky indukované   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The CB1 cannabinoid receptors have been found in the rodent suprachiasmatic nucleus, and their activation suppresses the light-induced phase shift in locomotor rhythmicity of mice and hamsters. Here, we show that the CB1 receptor agonist CP55940 significantly attenuates the light-induced phase delay in rats as well. Furthermore, it blocks the light induction of c-Fos and light-induced downregulation of pERK1/2 in the SCN, and the CB1 antagonist AM251 prevents the photic induction of pERK1/2 and reduces pGSK3β after photic stimulation. Our data suggest that the modulation of the cannabinoid receptor activity may affect the photic entrainment via the setting of the SCN sensitivity to light.

• MeSH
• agonisté kanabinoidních receptorů farmakologie   MeSH
• antagonisté kanabinoidních receptorů farmakologie   MeSH
• cyklohexanoly farmakologie   MeSH
• nucleus suprachiasmaticus účinky léků   fyziologie   účinky záření   MeSH
• piperidiny farmakologie   MeSH
• pohybová aktivita účinky léků   účinky záření   MeSH
• potkani Wistar MeSH
• pyrazoly farmakologie   MeSH
• světlo MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The adult circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus is resilient to glucocorticoids (GCs). The fetal rodent SCN resembles that of the adult in its organization of GC-sensitive peripheral tissues. We tested the hypothesis that the fetal SCN clock is sensitive to changes in GC levels. Maternal GCs must pass through the placenta to reach the fetal SCN. We show that the maternal but not the fetal part of the placenta harbors the autonomous circadian clock, which is reset by dexamethasone (DEX) and rhythmically expresses Hsd11b2. The results suggest the presence of a mechanism for rhythmic GC passage through the placental barrier, which is adjusted according to actual GC levels. GC receptors are expressed rhythmically in the laser-dissected fetal SCN samples. We demonstrate that hypothalamic explants containing the SCN of the mPer2 Luc mouse prepared at embryonic day (E)15 spontaneously develop rhythmicity within several days of culture, with dynamics varying among fetuses from the same litter. Culturing these explants in media enriched with DEX accelerates the development. At E17, treatment of the explants with DEX induces phase advances and phase delays of the rhythms depending on the timing of treatments, and the shifts are completely blocked by the GC receptor antagonist, mifepristone. The DEX-induced phase-response curve differs from that induced by the vehicle. The fetal SCN is sensitive to GCs in vivo because DEX administration to pregnant rats acutely downregulates c-fos expression specifically in the laser-dissected fetal SCN. Our results provide evidence that the rodent fetal SCN clock may respond to changes in GC levels.

• MeSH
• cirkadiánní hodiny účinky léků   genetika   fyziologie   MeSH
• cirkadiánní proteiny Period genetika   MeSH
• dexamethason farmakologie   MeSH
• glukokortikoidy farmakologie   fyziologie   MeSH
• hypothalamus fyziologie   MeSH
• krysa rodu rattus MeSH
• myši MeSH
• nucleus suprachiasmaticus účinky léků   fyziologie   MeSH
• placenta fyziologie   MeSH
• plod fyziologie   MeSH
• těhotenství MeSH
• vývoj plodu * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

AIM: The reactivity of the circadian clock in the suprachiasmatic nuclei (SCN) to stressful stimuli has been controversial but most studies have confirmed the resilience of the SCN to stress. We tested the hypothesis that during a critical period shortly after birth, the developing SCN clock is affected by glucocorticoids. METHODS: Mothers of 2 rat strains with different sensitivities to stress, that is Wistar rats and spontaneously hypertensive rats (SHR), and their pups were exposed to stressful stimuli every day from delivery, and clock gene expression profiles were detected in the 4-day-old pups' SCN. Levels of glucocorticoids in plasma were measured by LC-MS/MS. The glucocorticoid receptors antagonist mifepristone was administered to pups to block the effect of the glucocorticoids. RESULTS: The glucocorticoid receptors were detected at the mRNA and protein levels in the SCN of 4-day-old pups. The exposure of mothers to stressful stimuli elevated their plasma glucocorticoid levels. In Wistar rat pups, combination of daily maternal stress with their manipulation increased the plasma glucocorticoid levels and shifted the Bmal1 rhythm in the SCN which was completely blocked by mifepristone. In contrast, in SHR pups, maternal stress on its own caused phase shift of the Bmal1 expression rhythm in the SCN but the effect was mediated via glucocorticoid-independent mechanism. The Per1 and Per2 expression profiles remained phase-locked to the light/dark cycle. CONCLUSION: The results demonstrate that the SCN is sensitive to stressful stimuli early after birth in pups maintained under light/dark conditions and the effect is mediated via glucocorticoid-dependent pathways.

• MeSH
• antagonisté hormonů farmakologie   MeSH
• cirkadiánní hodiny * účinky léků   genetika   MeSH
• druhová specificita MeSH
• fotoperioda MeSH
• glukokortikoidy krev   MeSH
• laktace MeSH
• matka - expozice noxám MeSH
• mifepriston farmakologie   MeSH
• novorozená zvířata MeSH
• nucleus suprachiasmaticus účinky léků   metabolismus   patofyziologie   MeSH
• potkani inbrední SHR MeSH
• potkani Wistar MeSH
• psychický stres genetika   metabolismus   patofyziologie   MeSH
• receptory glukokortikoidů antagonisté a inhibitory   metabolismus   MeSH
• transkripční faktory ARNTL genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Signal transducers and activators of transcription (STAT) proteins regulate many aspects of cellular physiology from growth and differentiations to immune responses. Using immunohistochemistry, we show the daily rhythm of STAT3 protein in the rat suprachiasmatic nucleus (SCN), with low but significant amplitude peaking in the morning. We also reveal the strong expression of STAT5A in astrocytes of the SCN and the STAT5B signal in nonastrocytic cells. Administration of lipopolysaccharide (LPS) acutely induced phosphorylation of STAT3 on Tyr705 during both the day and the night and induced phosphorylation on Ser727 but only after the daytime application. The LPS-induced phospho-STAT3 (Tyr705) remained elevated for 24 hr after the daytime application but declined within 8 hr when LPS was applied at night.

• MeSH
• analýza rozptylu MeSH
• časové faktory MeSH
• cirkadiánní rytmus účinky léků   MeSH
• fosforylace účinky léků   MeSH
• gliový fibrilární kyselý protein metabolismus   MeSH
• krysa rodu rattus MeSH
• lipopolysacharidy farmakologie   MeSH
• nucleus suprachiasmaticus cytologie   účinky léků   MeSH
• potkani Wistar MeSH
• regulace genové exprese účinky léků   MeSH
• transkripční faktor STAT3 metabolismus   MeSH
• transkripční faktor STAT5 metabolismus   MeSH
• tyrosin-3-monooxygenasa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The circadian rhythms of many behavioral and physiological functions are regulated by the major circadian pacemaker in the suprachiasmatic nucleus. Long-term opiate addiction and drug withdrawal may affect circadian rhythmicity of various hormones or the sleep/activity pattern of many experimental subjects; however, limited research has been done on the long-term effects of sustained opiate administration on the intrinsic rhythmicity in the suprachiasmatic nucleus and pineal gland. Here we compared the effects of repeated daily treatment of rats with morphine or methadone and subsequent naloxone-precipitated withdrawal on the expression of the Per1, Per2, and Avp mRNAs in the suprachiasmatic nucleus and on arylalkylamine N-acetyltransferase activity in the pineal gland. We revealed that 10-day administration and withdrawal of both these drugs failed to affect clock genes and Avp expression in the SCN. Our results indicate that opioid-induced changes in behavioral and physiological rhythms originate in brain structures downstream of the suprachiasmatic nucleus regulatory output pathway. Furthermore, we observed that acute withdrawal from methadone markedly extended the period of high night AA-NAT activity in the pineal gland. This suggests that withdrawal from methadone, a widely used drug for the treatment of opioid dependence, may have stronger impact on melatonin synthesis than withdrawal from morphine.

• MeSH
• abstinenční syndrom metabolismus   MeSH
• arginin vasopresin metabolismus   MeSH
• cirkadiánní proteiny Period metabolismus   MeSH
• cirkadiánní rytmus účinky léků   MeSH
• hybridizace in situ MeSH
• methadon škodlivé účinky   MeSH
• morfin škodlivé účinky   MeSH
• narkotika škodlivé účinky   MeSH
• nucleus suprachiasmaticus účinky léků   metabolismus   MeSH
• potkani Wistar MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND AND PURPOSE: Opioids affect the circadian clock and may change the timing of many physiological processes. This study was undertaken to investigate the daily changes in sensitivity of the circadian pacemaker to an analgesic dose of morphine, and to uncover a possible interplay between circadian and opioid signalling. EXPERIMENTAL APPROACH: A time-dependent effect of morphine (1 mg·kg(-1) , i.p.) applied either during the day or during the early night was followed, and the levels of phosphorylated ERK1/2, GSK3β, c-Fos and Per genes were assessed by immunohistochemistry and in situ hybridization. The effect of morphine pretreatment on light-induced pERK and c-Fos was examined, and day/night difference in activity of opioid receptors was evaluated by [(35) S]-GTPγS binding assay. KEY RESULTS: Morphine stimulated a rise in pERK1/2 and pGSK3β levels in the suprachiasmatic nucleus (SCN) when applied during the day but significantly reduced both kinases when applied during the night. Morphine at night transiently induced Period1 but not Period2 in the SCN and did not attenuate the light-induced level of pERK1/2 and c-Fos in the SCN. The activity of all three principal opioid receptors was high during the day but decreased significantly at night, except for the δ receptor. Finally, we demonstrated daily profiles of pERK1/2 and pGSK3β levels in the rat ventrolateral and dorsomedial SCN. CONCLUSIONS AND IMPLICATIONS: Our data suggest that the phase-shifting effect of opioids may be mediated via post-translational modification of clock proteins by means of activated ERK1/2 and GSK3β.

• MeSH
• cirkadiánní hodiny účinky léků   MeSH
• cirkadiánní proteiny Period metabolismus   MeSH
• cirkadiánní rytmus účinky léků   MeSH
• fosforylace účinky léků   MeSH
• kinasa 3 glykogensynthasy metabolismus   MeSH
• krysa rodu rattus MeSH
• mitogenem aktivovaná proteinkinasa 1 metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 3 metabolismus   MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• morfin farmakologie   MeSH
• nucleus suprachiasmaticus účinky léků   metabolismus   MeSH
• potkani Wistar MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Most behavioral and physiological processes in living organisms exhibit periodic circadian rhythmicity. In mammals, these rhythms are coordinated by the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. In order to precisely synchronize free-running circadian oscillations to the 24h solar cycle, signals from the external environment, primarily the light/dark cycle, must reach the circadian clock within the SCN. A light pulse elevates intracellular Ca(2+) levels, and activates signaling cascades, leading to transcriptional activation of the clock genes mPer1 and mPer2 via phosphorylation of extracellular-signal-regulated kinases 1/2 (ERK1/2) and cyclic AMP-responsive element binding protein (CREB). Glutamate is the primary excitatory transmitter in retinal terminals in the SCN, and NMDA receptors (NMDAR) are the principal glutamate receptors that mediate the effect of light on resetting the circadian clock. Here we show the circadian rhythm in mRNA expression and protein level of the NMDAR 2B subunit (NR2B) in the SCN, with a peak at night. Also, we demonstrate ifenprodil inhibition of glutamate-induced phosphorylation of CREB (pCREB) and ERK1/2 (pERK1/2), and support thus the evidence for NR2B role in activation of signaling cascade involved in photic resetting of the circadian clock.

• MeSH
• antagonisté excitačních aminokyselin farmakologie   MeSH
• fosforylace MeSH
• hybridizace in situ MeSH
• krysa rodu rattus MeSH
• kyselina glutamová metabolismus   MeSH
• MAP kinasový signální systém MeSH
• nucleus suprachiasmaticus účinky léků   metabolismus   MeSH
• piperidiny farmakologie   MeSH
• potkani Wistar MeSH
• protein vázající cAMP responzivní element metabolismus   MeSH
• receptory N-methyl-D-aspartátu metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH