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.

Effects of 36 hours of sleep deprivation on military-related tasks: Can ammonium inhalants maintain performance?

INTRODUCTION: A lack of sleep can pose a risk during military operations due to the associated decreases in physical and cognitive performance. However, fast-acting ergogenic aids, such as ammonia inhalants (AI), may temporarily mitigate those adverse effects of total sleep deprivation (TSD). Therefore, the present study aimed to investigate the acute effect of AI on cognitive and physical performance throughout 36 hours of TSD in military personnel. METHODS: Eighteen male military cadets (24.1 ± 3.0 y; 79.3 ± 8.3 kg) performed 5 identical testing sessions during 36 hours of TSD (after 0 [0], 12 [-12], 24 [-24], and 36 [-36] hours of TSD), and after 8 [+8] hours of recovery sleep. During each testing session, the following assessments were conducted: Epworth sleepiness scale (ESS), simple reaction time (SRT), shooting accuracy (SA), rifle disassembling and reassembling (DAS), and countermovement jump height (JH). Heart rate (HR) was continuously monitored during the SA task, and a rating of perceived exertion (RPE) was obtained during the JH task. At each time point, tests were performed twice, either with AI or without AI as control (CON), in a counterbalanced order. RESULTS: There was faster SRT (1.6%; p < 0.01) without increasing the number of errors, higher JH (1.5%; p < 0.01), lower RPE (9.4%; p < 0.001), and higher HR (5.0%; p < 0.001) after using AI compared to CON regardless of TSD. However, neither SA nor DAS were affected by AI or TSD (p > 0.05). Independent of AI, the SRT was slower (3.2-9.3%; p < 0.001) in the mornings (-24, +8) than in the evening (-12), JH was higher (3.0-4.7%, p < 0.001) in the evenings (-12, -36) than in the mornings (0, -24, +8), and RPE was higher (20.0-40.1%; p < 0.001) in the sleep-deprived morning (-24) than all other timepoints (0, -12, -36, +8). Furthermore, higher ESS (59.5-193.4%; p < 0.001) was reported at -24 and -36 than the rest of the time points (0, -12, and + 8). CONCLUSION: Although there were detrimental effects of TSD, the usage of AI did not reduce those adverse effects. However, regardless of TSD, AI did result in a short-term increase in HR, improved SRT without affecting the number of errors, and improved JH while concurrently decreasing the RPE. No changes, yet, were observed in SA and DAS. These results suggest that AI could potentially be useful in some military scenarios, regardless of sleep deprivation.

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.

Underlying pharmacological mechanisms of psilocin-induced broadband desynchronization and disconnection of EEG in rats.

Introduction: Psilocybin is one of the most extensively studied psychedelic drugs with a broad therapeutic potential. Despite the fact that its psychoactivity is mainly attributed to the agonism at 5-HT2A receptors, it has high binding affinity also to 5-HT2C and 5-HT1A receptors and indirectly modulates the dopaminergic system. Psilocybin and its active metabolite psilocin, as well as other serotonergic psychedelics, induce broadband desynchronization and disconnection in EEG in humans as well as in animals. The contribution of serotonergic and dopaminergic mechanisms underlying these changes is not clear. The present study thus aims to elucidate the pharmacological mechanisms underlying psilocin-induced broadband desynchronization and disconnection in an animal model. Methods: Selective antagonists of serotonin receptors (5-HT1A WAY100635, 5-HT2A MDL100907, 5-HT2C SB242084) and antipsychotics haloperidol, a D2 antagonist, and clozapine, a mixed D2 and 5-HT receptor antagonist, were used in order to clarify the underlying pharmacology. Results: Psilocin-induced broadband decrease in the mean absolute EEG power was normalized by all antagonists and antipsychotics used within the frequency range 1-25 Hz; however, decreases in 25-40 Hz were influenced only by clozapine. Psilocin-induced decrease in global functional connectivity and, specifically, fronto-temporal disconnection were reversed by the 5-HT2A antagonist while other drugs had no effect. Discussion: These findings suggest the involvement of all three serotonergic receptors studied as well as the role of dopaminergic mechanisms in power spectra/current density with only the 5-HT2A receptor being effective in both studied metrics. This opens an important discussion on the role of other than 5-HT2A-dependent mechanisms underlying the neurobiology of psychedelics.

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.

Social defeat stress affects resident's clock gene and bdnf expression in the brain.

Social defeat stress affects behavior and changes the expression of the genes underlying neuronal plasticity in the brain. The circadian clock regulates most neuronal processes in the brain, which results in daily variations of complex behavior, and any disturbance in circadian clock oscillations increases the risk of mood and cognitive disbalance. In this study, we assessed the effect of acute and repeated social defeat stress on Per2 and Nr1d1 expression in prefrontal cortexes, hippocampi, pineal glands, olfactory bulbs, cerebella, and pituitary glands. We also evaluated the effect of our experimental setting on levels of Bdnf and plasma corticosterone, two markers widely used to asses the impact of stress on mammalian physiology. Our data show that single and repeated social defeat stress upregulates the expression of both clock genes and Bdnf in all brain structures, and corticosterone in the blood. While the general pattern of Bdnf upregulation suggests higher sensitivity in the intruder group, the clock genes are induced more significantly in residents, especially by repeated stress sessions. Our work thus suggests that the model of stress-induced anxiety and depression should consider a group of residents because, for some parameters, they may respond more distinctively than intruders.LAY SUMMARYThe resident/intruder experimental paradigm affects the expression of clock genes Per2, Nr1d1and Bdnf in the brain structures and plasma corticosterone level. The induction of clock genes is evident in both experimental groups; however, it is more marked in residents. Together with the significant increase in Bdnf levels in the majority of brain structures and plasma corticosterone in residents, our data suggest that in the model of social defeat stress, the utility of an experimental group of residents could be contributive.

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.

Neonatal Clonazepam Administration Induced Long-Lasting Changes in GABAA and GABAB Receptors.

Benzodiazepines (BZDs) are widely used in patients of all ages. Unlike adults, neonatal animals treated with BZDs exhibit a variety of behavioral deficits later in life; however, the mechanisms underlying these deficits are poorly understood. This study aims to examine whether administration of clonazepam (CZP; 1 mg/kg/day) in 7-11-day-old rats affects Gama aminobutyric acid (GABA)ergic receptors in both the short and long terms. Using RT-PCR and quantitative autoradiography, we examined the expression of the selected GABAA receptor subunits (α1, α2, α4, γ2, and δ) and the GABAB B2 subunit, and GABAA, benzodiazepine, and GABAB receptor binding 48 h, 1 week, and 2 months after treatment discontinuation. Within one week after CZP cessation, the expression of the α2 subunit was upregulated, whereas that of the δ subunit was downregulated in both the hippocampus and cortex. In the hippocampus, the α4 subunit was downregulated after the 2-month interval. Changes in receptor binding were highly dependent on the receptor type, the interval after treatment cessation, and the brain structure. GABAA receptor binding was increased in almost all of the brain structures after the 48-h interval. BZD-binding was decreased in many brain structures involved in the neuronal networks associated with emotional behavior, anxiety, and cognitive functions after the 2-month interval. Binding of the GABAB receptors changed depending on the interval and brain structure. Overall, the described changes may affect both synaptic development and functioning and may potentially cause behavioral impairment.

The day-night differences in ERK1/2, GSK3ß activity and c-Fos levels in the brain, and the responsiveness of various brain structures to morphine.

As with other drugs or pharmaceuticals, opioids differ in their rewarding or analgesic effects depending on when they are applied. In the previous study, we have demonstrated the day/night difference in the sensitivity of the major circadian clock in the suprachiasmatic nucleus to a low dose of morphine, and showed the bidirectional effect of morphine on pERK1/2 and pGSK3ß levels in the suprachiasmatic nucleus depending on the time of administration. The main aim of this study was to identify other brain structures that respond differently to morphine depending on the time of its administration. Using immunohistochemistry, we identified 44 structures that show time-of-day specific changes in c-Fos level and activity of ERK1/2 and GSK3ß kinases in response to a single dose of 1 mg/kg morphine. Furthermore, comparison among control groups revealed the differences in the spontaneous levels of all markers with a generally higher level during the night, that is, in the active phase of the day. We thus provide further evidence for diurnal variations in the activity of brain regions outside the suprachiasmatic nucleus indicated by the temporal changes in the molecular substrate. We suggest that these changes are responsible for generating diurnal variation in the reward behavior or analgesic effect of opioid administration.

The Effect of a Common Daily Schedule on Human Circadian Rhythms During the Polar Day in Svalbard: A Field Study.

All Arctic visitors have to deal with extreme conditions, including a constant high light intensity during the summer season or constant darkness during winter. The light/dark cycle serves as the most potent synchronizing signal for the biological clock, and any Arctic visitor attending those regions during winter or summer would struggle with the absence of those entraining signals. However, the inner clock can be synchronized by other zeitgebers such as physical activity, food intake, or social interactions. Here, we investigated the effect of the polar day on the circadian clock of 10 researchers attending the polar base station in the Svalbard region during the summer season. The data collected in Svalbard was compared with data obtained just before leaving for the expedition (in the Czech Republic 49.8175°N, 15.4730°E). To determine the circadian functions, we monitored activity/rest rhythm with wrist actigraphy followed by sleep diaries, melatonin rhythm in saliva, and clock gene expression (Per1, Bmal1, and Nr1D1) in buccal mucosa samples. Our data shows that the two-week stay in Svalbard delayed melatonin onset but did not affect its rhythmic secretion, and delayed the activity/rest rhythm. Furthermore, the clock gene expression displayed a higher amplitude in Svalbard compared to the amplitude detected in the Czech Republic. We hypothesize that the common daily schedule at the Svalbard expedition strengthens circadian rhythmicity even in conditions of compromised light/dark cycles. To our knowledge, this is the first study to demonstrate peripheral clock gene expression during a polar expedition.

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.

Neonatal Clonazepam Administration Induces Long-Lasting Changes in Glutamate Receptors.

γ-aminobutyric acid (GABA) pathways play an important role in neuronal circuitry formation during early postnatal development. Our previous studies revealed an increased risk for adverse neurodevelopmental consequences in animals exposed to benzodiazepines, which enhance GABA inhibition via GABAA receptors. We reported that administration of the benzodiazepine clonazepam (CZP) during postnatal days 7-11 resulted in permanent behavioral alterations. However, the mechanisms underlying these changes are unknown. We hypothesized that early CZP exposure modifies development of glutamatergic receptors and their composition due to the tight developmental link between GABAergic functions and maturation of glutamatergic signaling. These changes may alter excitatory synapses, as well as neuronal connectivity and function of the neural network. We used quantitative real-time PCR and quantitative autoradiography to examine changes in NMDA and AMPA receptor composition and binding in response to CZP (1 mg/kg/day) administration for five consecutive days, beginning on P7. Brains were collected 48 h, 1 week, or 60 days after treatment cessation, and mRNA subunit expression was assessed in the hippocampus and sensorimotor cortex. A separate group of animals was used to determine binding to NMDA in different brain regions. Patterns of CZP-induced alterations in subunit mRNA expression were dependent on brain structure, interval after CZP cessation, and receptor subunit type. In the hippocampus, upregulation of GluN1, GluN3, and GluR2 subunit mRNA was observed at the 48-h interval, and GluN2A and GluR1 mRNA expression levels were higher 1 week after CZP cessation compared to controls, while GluN2B was downregulated. CZP exposure increased GluN3 and GluR2 subunit mRNA expression levels in the sensorimotor cortex 48 h after treatment cessation. GluA3 was higher 1 week after the CZP exposure, and GluN2A and GluA4 mRNA were significantly upregulated 2 months later. Expression of other subunits was not significantly different from that of the controls. NMDA receptor binding increased 1 week after the end of exposure in most hippocampal and cortical areas, including the sensorimotor cortex at the 48-h interval. CZP exposure decreased NMDA receptor binding in most evaluated hippocampal and cortical areas 2 months after the end of administration. Overall, early CZP exposure likely results in long-term glutamatergic receptor modulation that may affect synaptic development and function, potentially causing behavioral impairment.

Circadian rhythms of melatonin and peripheral clock gene expression in idiopathic REM sleep behavior disorder.

OBJECTIVE: To evaluate changes in the expression of clock genes and melatonin levels in patients with idiopathic REM sleep behavior disorder (RBD) as a potential early stage of synucleinopathies. METHODS: We assessed the rhythmicity of circadian clock genes using real time-quantitative polymerase chain reaction and 24-h blood melatonin profiles using radio-immunoassay in 10 RBD patients and nine age-matched controls. RESULTS: The RBD patients did not show circadian rhythmicity for clock genes Per2, Bmal1, and Nr1d1 but the rhythmicity of Per 1 remained, and the amplitude of Per3 was diminished. The 24-h melatonin rhythm did not differ between RBD patients and healthy control subjects. Melatonin profile in RBD patients was delayed by 2 h compared to controls, the habitual sleep phases were phase delayed by about 1 h, however no phase shift occurred in any of the clock genes studied. The control group had stable acrophases of melatonin rhythms of approximately 5 h whereas the RBD patients had a more dispersed range over 11 h. CONCLUSIONS: Our results suggest that RBD could be associated with altered expression of clock genes and delayed melatonin secretion. Thus, we argue that circadian system dysregulation could play a role in RBD.