The role of N6-methyladenosine RNA methylation in the crosstalk of circadian clock and neuroinflammation in rodent suprachiasmatic nuclei.

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.

Impact of Prenatal LPS and Early-life Constant Light Exposure on Circadian Gene Expression Profiles in Various Rat Tissues.

Exposure to lipopolysaccharide (LPS) during prenatal development leads to various changes in neurobiological and behavioural patterns. Similarly, continuous exposure to constant light (LL) during the critical developmental period of the circadian system affects gene expression in various tissues in adulthood. Given the reciprocal nature of the interaction between the circadian and the immune systems, our study primarily investigated the individual effects of both interventions and, more importantly, their combined effect. We aimed to explore whether there might be a potential synergistic effect on circadian rhythms and their parameters, focussing on the expression of clock genes, immune-related genes, and specific genes in the hippocampus, pineal gland, spleen and adrenal gland of rats at postnatal day 30. Our results show a significant influence of prenatal LPS and postnatal LL on the expression profiles of all genes assessed. However, the combination of prenatal LPS and postnatal LL only revealed an enhanced negative effect in a minority of the comparisons. In most cases, it appeared to attenuate the changes induced by the individual interventions, restoring the measured parameters to values closer to those of the control group. In particular, genes such as Nr1d1, Aanat and Tph1 showed increased amplitude in the pineal gland and spleen, while the kynurenine enzymes Kynu and KatII developed circadian rhythmicity in the adrenal glands only after the combined interventions. Our data suggest that a mild immunological challenge during prenatal development may play a critical role in triggering an adaptive response of the circadian clock later in life.

The disruption of circadian rhythmicity of gene expression in the hippocampus and associated structures in Gria2R/R mice; a comparison with C57BL/6J and Adar2-/- mice strains.

Adar2-/- mice are a widely used model for studying the physiological consequences of reduced RNA editing. These mice are viable only when the Q/R editing site of the Gria2 subunit of the AMPA receptor is constitutively mutated to the codon for arginine, and Gria2R/R mice often serve as the sole control for Adar2-/- mice. Our study aimed to investigate whether ADAR2 inactivity and the Gria2R/R phenotype affect the rhythmicity of the circadian clock gene pattern and the expression of Gria1 and Gria2 subunits in the suprachiasmatic nucleus (SCN), hippocampus, parietal cortex and liver. Our data show that Gria2R/R mice completely lost circadian rhythmicity in the hippocampus compared to Adar2-/- mice. Compared to C57BL/6J mice, the expression profiles in the hippocampus and parietal cortex of Gria2R/R mice differ to the same extent as in Adar2-/-. No alterations were detected in the circadian profiles in the livers. These data suggest that the natural gradual postnatal increase in the editing of the Q/R site of the Gria2 subunit may be important for the development of circadian clockwork in some brain structures, and the use of Gria2R/R mice as the only control to Adar2-/- mice in the experiments dependent on the hippocampus and parietal cortex should therefore be considered.

Methadone administered to rat dams during pregnancy and lactation affects the circadian rhythms of their pups.

The circadian clock is one of the most important homeostatic systems regulating the majority of physiological functions. Its proper development contributes significantly to the maintenance of health in adulthood. Methadone is recommended for the treatment of opioid use disorders during pregnancy, increasing the number of children prenatally exposed to long-acting opioids. Although early-life opioid exposure has been studied for a number of behavioral and physiological changes observed later in life, information on the relationship between the effects of methadone exposure and circadian system development is lacking. Using a rat model, we investigated the effects of prenatal and early postnatal methadone administration on the maturation of the circadian clockwork in the suprachiasmatic nucleus (SCN) and liver, the rhythm of aralkylamine N-acetyltransferase (AA-NAT) activity in the pineal gland, and gene expression in the livers of 20-day-old rats. Our data show that repeated administration of methadone to pregnant and lactating mothers has significant effect on rhythmic gene expression in the SCN and livers and on the rhythm of AA-NAT in the offspring. Similar to previous studies with morphine, the rhythm amplitudes of the clock genes in the SCN and liver were unchanged or enhanced. However, six of seven specific genes in the liver showed significant downregulation of their expression, compared to the controls in at least one experimental group. Importantly, the amplitude of the AA-NAT rhythm was significantly reduced in all methadone-treated groups. As there is a strong correlation with melatonin levels, this result could be of importance for clinical practice.

Circadian control of kynurenine pathway enzymes in the rat pineal gland, liver, and heart and tissue- and enzyme-specific responses to lipopolysaccharide.

Amino acid tryptophan is catabolised via the kynurenine and serotonin-melatonin pathways, leading to various biologically active metabolites involved in regulating immunity, metabolism, and neuronal function. The levels of these metabolites are determined by the enzymes, which respond to altered homeostasis and pathological processes in the body. For the pineal gland, most work has centred on the serotonin-melatonin pathway. Still, no information exists on the expression of kynurenine pathway enzymes (KPEs), which may compete for the same substrate. Therefore, in this study, we investigated the physiological expression of KPEs in the rat pineal gland and their alterations in response to acute inflammation. We further compared the pineal expression profiles with the KPE expression in the rat liver and heart. Our data indicate the basal, non-induced expression of KPEs in the pineal gland, liver, and hearts, with a few first-step enzyme exceptions, such as Tdo and Ido1, and the first-step enzyme of serotonin pathway Tph1. This physiological expression was regulated in a circadian manner in the pineal gland and liver but not in the heart. Peripheral treatment with lipopolysaccharide resulted in mild upregulation of Tph1 in the pineal gland and heart, more robust upregulation of KPEs in the pineal gland and heart, but downregulation of Kmo, KatII, and Kynu in the liver. Altogether, our data provide evidence on the physiological expression of KPEs in the pineal gland, liver, and heart, which is regulated by the circadian clock in a tissue-specific manner. Furthermore, we show the temporal dynamics and bidirectional change in the transcriptional patterns of KPEs, Tph1, Per2, Nr1d1, and Stat3 in response to systemic administration of lipopolysaccharide in these tissues.

Circadian rhythms of locomotor activity in rats: Data on the effect of morphine administered from the early stages of embryonic development until weaning.

The circadian clock generates behavioural and physiological rhythms to maximize the efficacy of organismal functions. The circadian system with a major circadian pacemaker in the suprachiasmatic nucleus of the hypothalamus develops gradually and its proper function in adulthood depends on an appropriate neurochemical milieu during ontogeny [1]. Locomotor activity is under direct control by the circadian clock, and alterations in its rhythmicity indicate changes of circadian clock function. We evaluated circadian parameters of locomotor rhythms of adult male Wistar rats born to mothers that were exposed to a stable dose of 0.1 mg/ml of morphine in drinking water (36 ml water on average/day/each rat) from embryonic day 10 (E10) until weaning at postnatal day 28 (P28). Increasing the dose of morphine in drinking water was used to evaluate the changes in the rhythmic gene expression in the suprachiasmatic nucleus and in the livers of young rats at P20 [2]. At P90, we started measurement of endogenous rhythmicity for 12 days in constant darkness (DD), then we applied a 15 min light pulse at circadian time 15 (CT15) and followed the animals for the next 15 days in DD. We evaluated the magnitude of light-induced phase shift and compared the circadian parameters of free-running rhythmicity in the intervals before and after the light pulse. All data were also compared between morphine-exposed animals (M group) and controls (C group) that were not exposed to morphine. An unpaired t-test confirmed a significantly longer light-induced phase delay in M group compared with C group, a prolonged circadian period in M group in the interval after the light pulse, and greater amplitude for C group in the first interval, i.e. before the light pulse. No change in total activity counts between groups was confirmed.

The Circadian Rhythms of STAT3 in the Rat Pineal Gland and Its Involvement in Arylalkylamine-N-Acetyltransferase Regulation.

In rodents, the melatonin production by the pineal gland is controlled through adrenergic signaling from the suprachiasmatic nuclei and regulation of the principal enzyme in its synthesis, arylalkylamine-N-acetyltransferase (AANAT). In the present study, we identified increased isoprenaline-induced aa-nat expression and nocturnal AANAT activity in the pineal glands in response to the silencing of the signal transducer and activator of transcription 3 (STAT3) with siRNA or STAT3 inhibitors WP1066 and AZD1480. This AANAT activity enhancement in vivo did not interfere with light-induced AANAT suppression. Systemic or in vitro lipopolysaccharide (LPS) administration markedly increased Stat3 expression and STAT3 phosphorylation, but it did not significantly affect AANAT expression or activity. Simultaneous LPS administration and Stat3 silencing enhanced the aa-nat transcription and AANAT activity to a similar extent as Stat3 inhibition without LPS co-administration. Furthermore, we describe the circadian rhythmicity in Stat3 expression and the phosphorylated form of STAT3 protein in the rat pineal gland. Our data suggest that the higher nocturnal endogenous level of STAT3 in the pineal gland decelerates or hampers the process of NA-induced AANAT activation or affects the AANAT enzyme stability.

Maternal morphine intake during pregnancy and lactation affects the circadian clock of rat pups.

Early-life morphine exposure causes a variety of behavioural and physiological alterations observed later in life. In the present study, we investigated the effects of prenatal and early postnatal morphine on the maturation of the circadian clockwork in the suprachiasmatic nucleus and the liver, and the rhythm in aralkylamine N-acetyltransferase activity in the pineal gland. Our data suggest that the most affected animals were those born to control, untreated mothers and cross-fostered by morphine-exposed dams. These animals showed the highest mesor and amplitude in the rhythm of Per2, Nr1d1 but not Per1 gene expression in the suprachiasmatic nuclei (SCN) and arrhythmicity in AA-NAT activity in the pineal gland. In a similar pattern to the rhythm of Per2 expression in the SCN, they also expressed Per2 in a higher amplitude rhythm in the liver. Five of seven specific genes in the liver showed significant differences between groups in their expression. A comparison of mean relative mRNA levels suggests that this variability was caused mostly by cross-fostering, animals born to morphine-exposed dams that were cross-fostered by control mothers and vice versa differed from both groups of natural mothers raising offspring. Our data reveal that the circadian system responds to early-life morphine administration with significant changes in clock gene expression profiles both in the SCN and in the liver. The observed differences between the groups suggest that the dose, timing and accompanying stress events such as cross-fostering may play a role in the final magnitude of the physiological challenge that opioids bring to the developing circadian clock.

Diurnal and seasonal differences in cardiopulmonary response to exercise in morning and evening chronotypes.

Circadian clocks regulate multiple physiological domains from molecular to behavioral levels and adjust bodily physiology to seasonal changes in day length. Circadian regulation of cellular bioenergy and immunity in the cardiovascular and muscle systems may underpin the individual diurnal differences in performance capacity during exercise. Several studies have shown diurnal differences in cardiopulmonary parameters at maximal and submaximal workloads in morning and evening circadian human phenotypes. However, the effect of seasons on these changes was not elucidated. In this study, we recruited subjects with Morningness-Eveningness Questionnaire scores corresponding to morning and evening types. Subjects underwent morning (7:00-9:00) and evening (20:00-22:00) maximal workload spiroergometry in both winter and summer seasons. We analyzed their performance time, anaerobic threshold, heart rate, and respiratory parameters. Our results suggest that evening types manifest diurnal variations in physical performance, particularly in winter. They also have slower heart rate recovery than morning types, irrespective of the time of day or season. Compared to winter, the chronotype effect on the magnitude of morning-evening differences in performance time, maximal heart rate, and anaerobic threshold onset was more significant in summer. Our data are in concordance with previous observations and confirm the difference between morning and evening types in the timing of maximum performance capacity.

Circadian Regulation of GluA2 mRNA Processing in the Rat Suprachiasmatic Nucleus and Other Brain Structures.

The mammalian circadian system consists of a major circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus and peripheral clocks in the body, including brain structures. The SCN depends on glutamatergic neurotransmission for transmitting signals from the retina, and it exhibits spontaneous 24-h rhythmicity in neural activity. The aim of this work was to evaluate the degree and circadian rhythmicity of AMPA receptor GluA2 subunit R/G editing and alternative flip/flop splicing in the SCN and other brain structures in Wistar rats. Our data show that the circadian rhythmicity in the SCN's GluA2 mRNA level was highest at dawn, while the circadian rhythm in R/G editing peaked at CT10 and the rhythmic flip varied with the acrophase at the late subjective night. The circadian rhythmicity was confirmed for R/G editing and splicing in the CA3 hippocampal area, and rhythmic variation of the flip isoform was also measured in the olfactory bulbs and cerebellum. The correlations between the R/G editing and alternative flip/flop splicing revealed a structure-dependent direction. In the hippocampus, the edited (G)-form level was positively correlated with the flip variant abundance, in accord with published data; by contrast, in the SCN, the flip variant was in associated more with the unedited (R) form. The edited (G) form and flop isoform also predominated in the retina and cerebellum.

Constant Light in Critical Postnatal Days Affects Circadian Rhythms in Locomotion and Gene Expression in the Suprachiasmatic Nucleus, Retina, and Pineal Gland Later in Life.

The circadian clock regulates bodily rhythms by time cues that result from the integration of genetically encoded endogenous rhythms with external cycles, most potently with the light/dark cycle. Chronic exposure to constant light in adulthood disrupts circadian system function and can induce behavioral and physiological arrhythmicity with potential clinical consequences. Since the developing nervous system is particularly vulnerable to experiences during the critical period, we hypothesized that early-life circadian disruption would negatively impact the development of the circadian clock and its adult function. Newborn rats were subjected to a constant light of 16 lux from the day of birth through until postnatal day 20, and then they were housed in conditions of L12 h (16 lux): D12 h (darkness). The circadian period was measured by locomotor activity rhythm at postnatal day 60, and the rhythmic expressions of clock genes and tissue-specific genes were detected in the suprachiasmatic nuclei, retinas, and pineal glands at postnatal days 30 and 90. Our data show that early postnatal exposure to constant light leads to a prolonged endogenous period of locomotor activity rhythm and affects the rhythmic gene expression in all studied brain structures later in life.

Prenatal exposure to lipopolysaccharide induces changes in the circadian clock in the SCN and AA-NAT activity in the pineal gland.

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.

The effect of the cannabinoid receptor agonist and antagonist on the light-induced changes in the suprachiasmatic nucleus of rats.

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 pGSK3ß 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.

The day/night difference in the circadian clock's response to acute lipopolysaccharide and the rhythmic Stat3 expression in the rat suprachiasmatic nucleus.

The circadian clock in the suprachiasmatic nucleus (SCN) regulates daily rhythms in physiology and behaviour and is an important part of the mammalian homeostatic system. Previously, we have shown that systemic inflammatory stimulation with lipopolysaccharide (LPS) induced the daytime-dependent phosphorylation of STAT3 in the SCN. Here, we demonstrate the LPS-induced Stat3 mRNA expression in the SCN and show also the circadian rhythm in Stat3 expression in the SCN, with high levels during the day. Moreover, we examined the effects of LPS (1mg/kg), applied either during the day or the night, on the rhythm in locomotor activity of male Wistar rats. We observed that recovery of normal locomotor activity patterns took longer when the animals were injected during the night. The clock genes Per1, Per2 and Nr1d1, and phosphorylation of kinases ERK1/2 and GSK3ß are sensitive to external cues and function as the molecular entry for external signals into the circadian clockwork. We also studied the immediate changes in these clock genes expressions and the phosphorylation of ERK1/2 and GSK3ß in the suprachiasmatic nucleus in response to daytime or night-time inflammatory stimulation. We revealed mild and transient changes with respect to the controls. Our data stress the role of STAT3 in the circadian clock response to the LPS and provide further evidence of the interaction between the circadian clock and immune system.

Delayed Effect of the Light Pulse on Phosphorylated ERK1/2 and GSK3ß Kinases in the Ventrolateral Suprachiasmatic Nucleus of Rat.

The intrinsic period of circadian clock in the suprachiasmatic nucleus is entrained to a 24-h cycle by external cues, mainly light. Previous studies have shown that light applied at night induces robust phosphorylation of extracellular-signal-regulated kinase that is necessary to process the light pulse into the phase shift of the clock phase. In this study, we show the persistent downregulation of phosphorylated extracellular-signal-regulated kinase and transient downregulation of phosphorylated glycogen synthase kinase-3beta in the ventrolateral part of the suprachiasmatic nucleus to photic stimuli starting at 2 h after the beginning of the light pulse. As both kinases are involved in regulation of circadian clockwork, we hypothesize that these changes may contribute to the phase-shifting effect of light at night.