Chronic ethanol consumption impairs the circadian rhythm of pro-opiomelanocortin and period genes mRNA expression in the hypothalamus of the male rat

Certain psychiatric disorders are known to alter the body's biological rhythms. However, currently, very little information is known about the effect of chronic ethanol administration on the circadian clock or the rhythm of beta-endorphin-containing neurons that participate in the control of the reward and reinforcement of alcohol drinking. Here, we report that administration of ethanol, via a liquid diet paradigm for a period of 2 weeks, abolishes the circadian rhythm of pro-opiomelanocortin mRNA expression of beta-endorphin neurons in the arcuate nucleus of the hypothalamus. The circadian expression of the clock governing rat period genes (rPeriod1 mRNA and rPeriod2 mRNA) in the arcuate nucleus was significantly altered, suggesting that ethanol administration disrupted the internal clock. Moreover, ethanol consumption altered the circadian rhythms of rPeriod2 and rPeriod3 mRNA levels in the suprachiasmatic nucleus, suggesting that ethanol also affected the function of the central pacemaker. Our findings identified the vulnerability of the body's clock machinery and its opioidergic system to chronic alcohol drinking.     

Circadian expression of clock genes in the rat eye and brain

The light sensing system in the eye directly affects the circadian oscillator in the mammalian suprachiasmatic nucleus (SCN). To investigate this relationship in the rat, we examined the circadian expression of clock genes in the SCN and eye tissue during a 24 h day/night cycle. In the SCN, rPer1 and rPer2 mRNAs were expressed in a clear circadian rhythm like rCry1 and rCry2 mRNAs, whereas the level of BMAL1 and CLOCK mRNAs decreased during the day and increased during the night with a relatively low amplitude. It seems that the clock genes of the SCN may function in response to a master clock oscillation in the rat. In the eye, the rCry1 and rCry2 were expressed in a circadian rhythm with an increase during subjective day and a decrease during subjective night. However, the expression of Opn4 mRNA did not exhibit a clear circadian pattern, although its expression was higher in daytime than at night. This suggests that cryptochromes located in the eye, rather than melanopsin, are the major photoreceptive system for synchronizing the circadian rhythm of the SCN in the rat.     

Diurnal rhythmic expression of the rhythm-related genes, <Emphasis Type="Italic">rPeriod1, rPeriod2,</Emphasis> and <Emphasis Type="Italic"> rClock</Emphasis>, in the rat brain

High densities of the mRNA of three rhythm-related genes, rPeriod1 (rPer1), rPer2, and rClock, which share high homology in Drosophila and mammals, are found in the rat hypothalamic suprachiasmatic nucleus (SCN). The SCN, however, is not the only brain region that expresses these genes. To understand the possible physiological roles of these rhythm-related genes, we examined expression of these genes in different brain regions at various time points in male Sprague--Dawley rats. Using semi quantitativein situ hybridization with ³⁵S-riboprobes to evaluate mRNA levels, the diurnal rhythmicity of rPer1, and rPer2 mRNA levels was found in the SCN, arcuate nucleus, and median eminence/pars tuberalis. Expression patterns of mRNA for rPer1 and rPer2, however, were not similar in these brain regions. The rhythmicity in these brain regions was specific, because it was not observed in the cerebellum or hippocampus. Moreover, diurnal changes in rClock mRNA expression were not detected in any of the brain regions examined. These findings suggest that the different expression patterns observed for rPer1, rPer2, and rClock mRNAs may be attributed to their different physiological roles in these brain regions, and support previous work indicating that circadian rhythms in the brain are widespread.     

Timed maternal melatonin treatment reverses circadian disruption of the fetal adrenal clock imposed by exposure to constant light

Surprisingly, in our modern 24/7 society, there is scant information on the impact of developmental chronodisruption like the one experienced by shift worker pregnant women on fetal and postnatal physiology. There are important differences between the maternal and fetal circadian systems; for instance, the suprachiasmatic nucleus is the master clock in the mother but not in the fetus. Despite this, several tissues/organs display circadian oscillations in the fetus. Our hypothesis is that the maternal plasma melatonin rhythm drives the fetal circadian system, which in turn relies this information to other fetal tissues through corticosterone rhythmic signaling. The present data show that suppression of the maternal plasma melatonin circadian rhythm, secondary to exposure of pregnant rats to constant light along the second half of gestation, had several effects on fetal development. First, it induced intrauterine growth retardation. Second, in the fetal adrenal in vivo it markedly affected the mRNA expression level of clock genes and clock-controlled genes as well as it lowered the content and precluded the rhythm of corticosterone. Third, an altered in vitro fetal adrenal response to ACTH of both, corticosterone production and relative expression of clock genes and steroidogenic genes was observed. All these changes were reversed when the mother received a daily dose of melatonin during the subjective night; supporting a role of melatonin on overall fetal development and pointing to it as a 'time giver' for the fetal adrenal gland. Thus, the present results collectively support that the maternal circadian rhythm of melatonin is a key signal for the generation and/or synchronization of the circadian rhythms in the fetal adrenal gland. In turn, low levels and lack of a circadian rhythm of fetal corticosterone may be responsible of fetal growth restriction; potentially inducing long term effects in the offspring, possibility that warrants further research.     

Identification and functional analysis of a novel ligand for G protein-coupled receptor, Neuromedin S

We identified a novel 36-amino acid neuropeptide in rat brain as an endogenous ligand for the G protein-coupled receptors FM-3/GPR66 and FM-4/TGR-1, which were identified to date as the neuromedin U (NMU) receptors, and designated this peptide neuromedin S (NMS) because it was specifically expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. NMS shared a C-terminal core structure with NMU. NMS mRNA was highly expressed in the central nervous system, spleen and testis. In rat brain, NMS expression was restricted to the ventrolateral portion of the SCN and has a diurnal peak under light/dark cycling, but remains stable under constant darkness. Intracerebroventricular (ICV) administration of NMS in rats induced nonphotic type phase shifts in the circadian rhythm of locomotor activity. ICV injection of NMS also decreased 12-h food intake during the dark period in rats. This anorexigenic effect was more potent than that observed with the same dose of NMU. ICV administration of NMS increased proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus (Arc) and corticotropin-releasing hormone mRNA in the paraventricular nucleus, and induced c-Fos expression in the POMC neurons in the Arc. These findings suggest that NMS is implicated in the regulation of circadian rhythm and feeding behavior.     

Steroid control of gonadotropin-releasing hormone secretion: associated changes in pro-opiomelanocortin and preproenkephalin messenger RNA expression in the ovine hypothalamus

The endogenous opioid peptides have been implicated in mediating the actions of estrogen and progesterone on GnRH release. We used in situ hybridization histochemistry to determine whether steroid-induced changes in GnRH/LH release in the female sheep are associated with changes in the cellular mRNA content of the precursors for beta-endorphin (pro-opiomelanocortin; POMC) and met-enkephalin (pre-proenkephalin; PENK). Two specific hypotheses were tested. First, that the inhibitory actions of progesterone are associated with an increase in opioid gene expression in specific hypothalamic nuclei. Our data support this hypothesis. Thus, an increase in progesterone was associated with increased POMC gene expression in the arcuate nucleus and PENK in the paraventricular nucleus. Further, the increase in POMC was restricted to regions of the arcuate nucleus that contain steroid sensitive beta-endorphin neurons. Our second hypothesis, that gene expression for the two opioid precursors would decrease prior to the start of the estradiol-stimulated GnRH surge, was not supported. Rather, POMC (but not PENK) gene expression in the arcuate nucleus was significantly higher in estradiol-treated animals than controls at the peak of the GnRH surge. These data suggest that beta-endorphin neurons in subdivisions of the arcuate nucleus and enkephalin neurons in the paraventricular nucleus are part of the neural network by which progesterone inhibits LH release. While enkephalin neurons may not play a role in estrogen positive feedback, increases in POMC mRNA in the arcuate nucleus at the time of the GnRH peak may be important for replenishing beta-endorphin stores and terminating estrous behavior.     

Effect of long-term exposure to constant dim light on the circadian system of rats

The newly discovered multi-oscillatory nature of the mammalian circadian clock system and the cloning of the genes involved in the molecular mechanism that generates circadian rhythmicity have opened new approaches for understanding how mammals are temporally organized and how the mammalian circadian system reacts to the lack of normal synchronization cues. In the present study we investigated the effects of long-term exposure to constant red dim light on the pattern of the expression of Period 1 in the suprachiasmatic nuclei of the hypothalamus and of Arylalkylamine N-acetyltransferase(Aa-nat) in the retina and pineal gland. Our data demonstrate that Period 1 mRNA expression in the suprachiasmatic nuclei of the hypothalamus was not affected by exposure to constant red dim light for 60 days, whereas Aa-nat mRNA expression in the retina and in the pineal gland was significantly affected, since in some animals (20-30%) Aa-nat mRNA levels were found to be higher during the subjective day. A circadian rhythm of serum melatonin and locomotor activity was present in all the animals tested. In 4 animals serum melatonin levels were high during the subjective day. Our data suggest that long-term exposure to constant red dim light may induce desynchronization between the circadian rhythm of locomotor activity and serum melatonin levels.     

Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (α-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-α-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POMC mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 µg/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POMC neurons in the hypothalamus and an increased mRNA expression of these neuropeptides.     

MSG effects on beta-endorphin and alpha-MSH in the hypothalamus and caudal medulla

Monosodium glutamate (MSG) was given to neonatal male rats to determine its effects on neurons containing beta-endorphin (beta-END) and alpha-melanocyte stimulating hormone (alpha-MSH) within the basal hypothalamus (arcuate nucleus) and caudal medulla [nucleus tractus solitarius (NTS)] and on the levels of beta-END and alpha-MSH within these areas. Immunocytochemical studies demonstrated a reduction in the number of cells within the medial hypothalamic area (arcuate nucleus) among MSG-treated animals versus saline controls. MSG did not reduce the number of cell bodies within the caudal medulla (NTS). MSG significantly reduced beta-END and alpha-MSH immunoreactive levels in the basal hypothalamus as determined by radioimmunoassay. Whereas a significant reduction in the level of beta-END occurred in the ventral caudal medulla (VCM), none occurred in the dorsal caudal medulla (DCM). In contrast, levels of alpha-MSH increased significantly in the DCM among animals receiving MSG compared to control animals. This study documents the contribution of beta-endorphin containing neurons of the basal hypothalamus to areas of the caudal medulla. The effect of MSG on beta-endorphin and alpha-MSH neurons in these areas and their differential effects on levels in the caudal medulla areas raises questions about the sites of origin of these peptides.     

Glucocorticoids and Stress-Induced Changes in the Expression of PERIOD1 in the Rat Forebrain

The secretion of glucocorticoids in mammals is under circadian control, but glucocorticoids themselves are also implicated in modulating circadian clock gene expression. We have shown that the expression of the circadian clock protein PER1 in the forebrain is modulated by stress, and that this effect is associated with changes in plasma corticosterone levels, suggesting a possible role for glucocorticoids in the mediation of stress-induced changes in the expression of PER1 in the brain. To study this, we assessed the effects of adrenalectomy and of pretreatment with the glucocorticoid receptor antagonist, mifepristone, on the expression of PER1 in select limbic and hypothalamic regions following acute exposure to a neurogenic stressor, restraint, or a systemic stressor, 2-Deoxy-D-glucose (2DG) in rats. Acute restraint suppressed PER1 expression in the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and the central nucleus of the amygdala (CEAl), whereas 2DG increased PER1 in both regions. Both stressors increased PER1 expression in the paraventricular (PVN) and dorsomedial (DMH) nuclei of the hypothalamus, and the piriform cortex (Pi). Adrenalectomy and pretreatment with mifepristone reversed the effects of both stressors on PER1 expression in the BNSTov and CEAl, and blocked their effects in the DMH. In contrast, both treatments enhanced the effects of restraint and 2DG on PER1 levels in the PVN. Stress-induced PER1 expression in the Pi was unaffected by either treatment. PER1 expression in the suprachiasmatic nucleus, the master circadian clock, was not altered by either exposure to stress or by the glucocorticoid manipulations. Together, the results demonstrate a key role for glucocorticoid signaling in stress-induced changes in PER1 expression in the brain.     

Establishment of cell lines derived from the rat suprachiasmatic nucleus

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.     

Leptin sensitive neurons in the hypothalamus.

A major paradigm in the field of obesity research is the existence of an adipose tissue-brain endocrine axis for the regulation of body weight. Leptin, the peptide mediator of this axis, is secreted by adipose cells. It lowers food intake and body weight by acting in the hypothalamus, a region expressing an abundance of leptin receptors and a variety of neuropeptides that influence food intake and energy balance. Among the most promising candidates for leptin-sensitive cells in the hypothalamus are arcuate nucleus neurons that co-express the anabolic neuropeptides, neuropeptide Y (NPY) and agouti-related peptide (AGRP), and those that express proopiomelanocortin (POMC), the precursor of the catabolic peptide, alphaMSH. These cell types contain mRNA encoding leptin receptors and show changes in neuropeptide gene expression in response to changes in food intake and circulating leptin levels. Decreased leptin signaling in the arcuate nucleus is hypothesized to increase the expression of NPY and AGRP. Levels of leptin receptor mRNA and leptin binding are increased in the arcuate nucleus during fasting, principally in NPY/AGRP neurons. These findings suggest that changes in leptin receptor expression in the arcuate nucleus are inversely associated with changes in leptin signaling, and that the arcuate nucleus is an important target of leptin action in the brain.     

Postnatal ontogenesis of the circadian clock within the rat liver

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

Constant light exposure aggravates POMC-mediated muscle wasting associated with hypothalamic alteration of circadian clock and SIRT1 in endotoxemia rats

Constant light exposure is widespread in the intensive care unit (ICU) and could increase the rate of brain dysfunction as delirium and sleep disorders in critical patients. And the activation of hypothalamic neuropeptides is proved to play a crucial role in regulating hypercatabolism, especially skeletal muscle wasting in critical patients, which could lead to serious complications and poor prognosis. Here we investigated the hypothesis that constant light exposure could aggravate skeletal muscle wasting in endotoxemia rats and whether it was associated with alterations of circadian clock and hypothalamic proopiomelanocortin(POMC) expression. Fifty-four adult male Sprague-Dawley rats were intraperitoneally injected with lipopolysaccharide(LPS) or saline, subjected to constant light or a 12:12?h light-dark cycle for 7 days. On day 8, rats were sacrificed across six time points in 24?h and hypothalamus tissues and skeletal muscle were obtained. Rates of muscle wasting were measured by 3-methylhistidine(3-MH) and tyrosine release as well as expression of two muscle atrophic genes, muscle ring finger 1(MuRF-1) and muscle atrophy F-box(MAFbx). The expression of circadian clock genes, silent information regulator 1(SIRT1), POMC and hypothalamic inflammatory cytokines were also detected. Results showed that LPS administration significantly increased hypothalamic POMC expression, inflammatory cytokine levels and muscle wasting rates. Meanwhile constant light exposure disrupted the circadian rhythm, declined the expression of SIRT1 as well as aggravated hypothalamic POMC overexpression and skeletal muscle wasting in rats with endotoxemia. Taken together, the results demonstrated that constant light exposure could aggravate POMC-mediated skeletal muscle wasting in endotoxemia rats, which is associated with alteration of circadian clocks and SIRT1 in the hypothalamus.