Search

Mice lacking the farnesoid X receptor (FXR) are used as a genetic model for nonalcoholic fatty liver disease because their livers exhibit hepatic steatosis and inflammation. The influence of taurine drinking on disrupted hepatic function was investigated using female Fxr-null mice. Significant decreases in the levels of hepatic damage-associated diagnostic markers, hepatic triglycerides, non-esterified fatty acids, and total bile acids were found in Fxr-null mice that had drunk water containing 0.5% taurine for four weeks. Hepatic but not serum taurine concentrations were significantly increased in these mice. The expression levels of oxidative stress-related genes (Hmox1 and Gsta1) and fatty acid synthetic genes (Acc1 and Scd1) were significantly decreased in these mice. These results suggest that drinking taurine improves hepatic steatosis and dysfunction caused by a lack of FXR.

Circadian rhythm is a fundamental biological system involved in the regulation of various physiological functions. However, little is known about a nature or function of circadian clock in human primary cells. In the present study, we have applied in vitro real time circadian rhythm monitoring to study human clock properties using primary skin fibroblasts. Among factors that affect human physiology, slightly lower extracellular pH was chosen to test its effects on circadian rhythm expression. We established human primary fibroblast cultures obtained from three healthy subjects, stably delivered a circadian reporter gene Bmal1-luciferase, and recorded circadian rhythms in the culture medium at pH 7.2 and pH 6.7. At pH 7.2, robust and sustained circadian rhythms were observed with average period length 24.47 ± 0.03 h. Such rhythms were also found at pH 6.7; however, period length was significantly shortened to 22.60 ± 0.20, amplitude was increased, and damping rate was decreased. The effect of exposure to low pH on the period length was reversible. The shortened period was unlikely caused by factors affecting cell viability because cell morphology and MTT assay showed no significant difference between the two conditions. In summary, our results showed that the circadian rhythm expression is affected at pH 6.7 in human primary fibroblasts without affecting cell viability.

Alterations in circadian rhythm generation may be related to the development of mood disorders. Although it has been reported that the most popular antidepressant, selective serotonin reuptake inhibitors (SSRIs) affect circadian phase, no data are available that describe the effects of SSRIs on other circadian parameters (period, amplitude and damping rate) in dissociated cells. In the present study we used real-time monitoring of bioluminescence in rat-1 fibroblasts expressing the Period1-luciferase transgene, and that in Period1-luciferase transgenic mouse suprachiasmatic nucleus (SCN) explants, in order to characterize the effects of SSRI on circadian oscillator function in vitro. We found that mRNA of the serotonin transporter (SERT), a target of SSRIs, was expressed in rat-1 fibroblasts. Sertraline, fluoxetine, fluvoxamine, citalopram and paroxetine all significantly shortened the period of Period1-bioluminescence rhythms in rat-1 fibroblasts. The amplitude was reduced by sertraline, and the damping rate was decreased by sertraline, fluoxetine, flvoxamine and paroxetine. The effect of sertraline was dose-dependent, and it also shortened the circadian period in the SCN. SERT is associated with lipid microdomains, which are required for efficient SERT activity. Indeed, cholesterol chelating reagent methyl-beta-cyclodextrin significantly reduced the period and the amplitude in rat-1 fibroblasts. Furthermore, lipid binding reagent xylazine significantly reduced the period. In summary our data present evidence that SSRIs affect circadian rhythmicity. The action of SSRIs is likely mediated by suppression of SERT activity. A better understanding of the relationship between mental illness and biological timing may yield new insight into disease etiology and avenues for treatment.

The mammalian pineal gland is an important component of the circadian system. In the present study, we examined the expression of roughly 8000 genes in the rat pineal gland as a function of time of day under light-dark (LD) cycles and in constant dark (DD) using oligo DNA microarray technique. We identified 47 and 13 genes that showed higher levels at night and day, respectively, under LD. The same patterns of expression were also observed in DD. About half of the genes that peaked at night have a known biological function, i.e., transcription factors and proteins that are involved in signaling cascades, whereas 14 are expressed sequence tags and 8 have an unknown biological function. Twelve of the genes that are up-regulated at night were also up-regulated after 1 h NE stimulation, thus suggesting that the expression of these genes is controlled by adrenergic mechanisms. Of the 13 genes that were up-regulated in the daytime, 6 coded for proteins that are involved in intracellular signaling pathways. The results obtained with microarray analysis were well correlated with data obtained using real time quantitative RT-PCR. The present results provide new materials to dissect and understand the pineal physiology.

Several studies have demonstrated that the mammalian retina contains an autonomous circadian clock. Dopaminergic and other inner retinal neurons express many of the clock genes, whereas some of these genes seem to be absent from the photoreceptors. This observation has led to the suggestion that in mammalian retina the circadian pacemaker driving retinal rhythms is located in the inner nuclear layer. However, other evidence points to the photoreceptor layer as the site of the mammalian retinal clock. The goal of the present study was to demonstrate the presence of a functional circadian clock in photoreceptors. First, using laser capture microdissection and reverse transcriptase-polymerase chain reaction, we investigated which of the clock genes are expressed in rat photoreceptors. We then prepared photoreceptor layer cultures from the retina to test whether these isolated cultures were viable and could drive circadian rhythms. Our data indicated that Per1, Per3, Cry1, Cry2, Clock, Bmal1, Rev-erb alpha, and Rora RNAs were present in the photoreceptors, whereas we were unable to amplify mRNA for Per2 and Npas2. Photoreceptor layers obtained from Period1-luciferase rats expressed a robust circadian rhythm in bioluminescence and melatonin synthesis. These results demonstrate that mammalian photoreceptors contain the circadian pacemaker driving rhythmic melatonin synthesis.

Circadian rhythms are endogenous cycles with periods close to, but not exactly equal to, 24 h. In mammals, circadian rhythms are generated in the suprachiasmatic nucleus (SCN) of the hypothalamus as well as several peripheral cell types, such as fibroblasts. Protein kinases are key regulators of the circadian molecular machinery. We investigated the role of the c-Jun N-terminal kinases (JNK), which belong to the mitogen-activated protein kinases family, in the regulation of circadian rhythms. In rat-1 fibroblasts, the p46 kDa, but not the p54 kDa, isoforms of JNK expressed circadian rhythms in phosphorylation. The JNK-inhibitor SP600125 dose-dependently extended the period of Period1-luciferase rhythms in rat-1 fibroblasts from 24.23+/-0.17-31.48+/-0.07 h. This treatment also dose-dependently delayed the onset of the bioluminescence rhythms. The effects of SP600125 on explant cultures from Period1-luciferase transgenic mice and Period2(Luciferase) knockin mice appeared tissue-specific. SP600125 lengthened the period in SCN, pineal gland, and lung explants in Period1-luciferase and Period2(Luciferase) mice. However, in the kidneys circadian rhythms were abolished in Period1-luciferase, while circadian rhythms were not affected by SP600125 treatment in Period2(Luciferase) mice. Valproic acid, already known to affect period length, enhanced JNK phosphorylation and, as predicted, shortened the period of the Period1-bioluminescence rhythms in rat-1 fibroblasts. In conclusion, our results showed that SP600125 treatment, as well as valproic acid, alters JNK phosphorylation levels, and modulates the period length in various tissues. We conclude that JNK phosphorylation levels may help to set the period length of mammalian circadian rhythms.

We examined Period (Per) mRNA rhythms in the suprachiasmatic nucleus (SCN) of a diurnal rodent and assessed how phase-shifting stimuli acutely affect SCN Per mRNA using semiquantitative in situ hybridization. First, Per1 and Per2 varied rhythmically in the SCN over the course of one circadian cycle in constant darkness: Per1 mRNA was highest in the early to mid-subjective day, while Per2 mRNA levels peaked in the late subjective day. Second, acute light exposure in the early subjective night significantly increased both Per1 and Per2 mRNA. Third, Per2 but not Per1 levels decreased 1 and 2 h after injection of the gamma-aminobutyric acid (GABA)(A) receptor agonist muscimol into the SCN during the subjective day. Fourth, muscimol also reduced the light-induced Per2 in the early subjective night, but Per1 induction by light was not significantly affected. Consistent with previous studies, these data demonstrate that diurnal and nocturnal animals show very similar daily patterns of Per mRNA and light-induced Per increases in the SCN. As with light, muscimol alters circadian phase, and daytime phase alterations induced by muscimol are associated with significant decreases in Per2 mRNA. In diurnal animals, muscimol-induced decreases in Per are associated with phase delays rather than advances. The direction of the daytime phase shift may be determined by the relative suppression of Per1 vs. Per2 in SCN cells. As in nocturnal animals, changes in Per1 and Per2 mRNA by photic and non-photic stimuli appear to be associated with circadian phase alteration.

Melatonin and its autonomic regulation serve important physiological functions. We recently demonstrated that stimulation of beta-adrenergic receptors only increases nighttime arylalkylamine N-acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) mRNA levels in mouse pineal gland in vitro, which suggests that pineal clocks may gate Aa-Nat gene expression. In the present study, our data reveal that cAMP analog increased Aa-Nat at any time of day but only in the presence of ionomycin. Using Fura-2AM in ratiometric calcium measurements, we show that isoproterenol stimulation increased intracellular free calcium levels at night, contrary to previous reports. Further, intra- or extracellular calcium depletion suppressed the isoproterenol-induced calcium responses as well as Aa-Nat gene expression. These results suggest calcium may be a critical factor in isoproterenol-induced Aa-Nat gene expression, which may be limited in the daytime. We also found that basal intracellular calcium levels were lower during the night and responses to isoproterenol and KCl depolarization were more robust. In addition, pineals of Cryptochrome mutant mice exhibited no significant difference between day and nighttime basal calcium or isoproterenol response. Together, these results suggest that basal calcium levels in the pineal may be controlled by the endogenous pineal clock, which may influence calcium dynamics, cellular homeostasis and sensitivity to external stimulation. Although the mechanism underlying Aa-Nat gene expression has been well studied, the role of calcium as a link between the pineal clock and Aa-Nat gene expression has been underestimated in rodent pineals.

In mammals it has been thought that the circadian clock localizes only in the suprachiasmatic nucleus of the hypothalamus. Recent studies have revealed that certain brain regions and peripheral tissues may also have intrinsic circadian clocks. However, the roles played by 'peripheral circadian clocks' have not been fully elucidated. In this study, we investigated their function using mouse pineal glands, and found that expression of the arylalkylamine N-acetyltransferase (Aa-Nat, EC 2.3.1.87, the rate-limiting enzyme of melatonin synthesis) gene after adrenergic receptor stimulation depended on the time of day even in vitro (gating). Phase-dependent Aa-Nat responses were observed in both melatonin-proficient and melatonin-deficient mouse pineal glands. Phosphodiesterases are unlikely to suppress Aa-Nat induction because a phosphodiesterase inhibitor itself had no effect on the mRNA levels. Puromycin was ineffective in inducing Aa-Nat mRNA levels in either the presence or absence of isoproterenol, suggesting that newly synthesized proteins may not be necessary to gate the Aa-Nat responses. We also discovered circadian dependence of the expression of Period1-luminescence in Period1-luciferase transgenic mouse pineal glands: circadian clocks may be functional in culture. Aa-Nat mRNA levels showed no significant circadian rhythms in the absence of isoproterenol, thus suggesting that Aa-Nat mRNA levels are induced by adrenergic mechanisms, not by a pineal circadian clock. Our results suggest that the pineal circadian clock may determine timing when Aa-Nat gene expression can respond to inputs from the master circadian clock in the suprachiasmatic nucleus, e.g. adrenergic stimulation.

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.

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains a central circadian pacemaker, which adjusts circadian rhythms within the body to environmental light–dark cycles. It has been shown that dark exposure in the day causes phase shifts in circadian rhythms, but it does not induce changes in the melatonin levels in the pineal gland. In this study, we examined the effect of dark exposure on two “circadian clock” genes Period1 and Period2 mRNA levels in the rat SCN, and on Period1, Period2, and arylalkylamine N-acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) gene expression in the pineal gland. Period1 and Period2 mRNA levels were significantly decreased in the SCN after 0.5 and 2 h, respectively, therefore suggesting that changes in those mRNA levels may be the part of the mechanisms of dark-induced phase shifts. Period1 and Aa-Nat mRNA levels in the pineal gland were not affected by darkness, but Period2 was moderately affected. Since Period1 and Aa-Nat mRNA levels in the pineal gland did not respond to dark stimulation, we further examined whether the pineal gland itself is capable of responding to adrenergic stimulation at this time of the day. Isoproterenol significantly induced Period1 and Aa-Nat mRNA levels; however, it did not affect Period2. Although previous studies have reported that during the day the SCN “gates” the dark information reaching the pineal, our data demonstrate that dark information may reach the pineal during the daytime.

Tryptophan hydroxylase is the first of four enzymes in the melatonin biosynthetic pathway. Recent studies have shown that there are two genes, Tph1 and Tph2, that encode tryptophan hydroxylase in mammals. In this study, we investigated which of the two genes is expressed in the rat retina. To that end, we measured Tph1 (classical Tph) and Tph2 mRNA levels using real-time quantitative RT-PCR in the retina. Our data demonstrate that Tph1 mRNA is the prevalent form expressed in the retina; Tph2 mRNA is also present but the level is very low. We also measured Tph1 expression levels in the outer nuclear layer, inner nuclear layer, and ganglion cell layer by combining laser capture microdissection and real-time RT-PCR. Tph1 mRNA is more abundant in the photoreceptors of the outer nuclear layer than in the inner nuclear layer or ganglion cell layer. Tph1 and Tph2 transcripts showed robust diurnal rhythms of abundance, with highest levels at night. Our results support the hypothesis that Tph1 is involved in melatonin synthesis in retinal photoreceptor cells.

Melatonin is synthesized in retinal photoreceptor cells and acts as a neuromodulator imparting photoperiodic information to the retina. The synthesis of melatonin is controlled by an ocular circadian clock and by light in a finely tuned mechanism that ensures that melatonin is synthesized and acts only at night in darkness. Here we report that the circadian clock gates melatonin synthesis in part by regulating the expression of the type 1 adenylyl cyclase (AC1) and the synthesis of cAMP in photoreceptor cells. This gating is effected through E-box-mediated transcriptional activation of the AC1 gene, which undergoes robust daily fluctuations that persist in constant illumination. The circadian control of the cAMP signaling cascade indicates that the clock has a more general and profound impact on retinal functions than previously thought. In addition, rhythmic control of AC1 expression was observed in other parts of the central circadian axis, the suprachiasmatic nucleus and pineal gland, but not in other brain areas examined. Thus, clock control of the cAMP signaling cascade may play a central role in the integration of circadian signals that control physiology and behavior.

Arylalkylamine N-acetyltransferase (AA-NAT) is the key regulatory enzyme in the melatonin biosynthetic pathway. Previous investigations have reported that Aa-nat mRNA in rat is only detected in a sub-population of photoreceptor cells that resemble cones in shape and size. In the present study, we investigated Aa-nat expression in the rat retina by using in situ hybridization and laser capture microdissection combined with the reverse transcription/polymerase chain reaction technique. Our results demonstrate that, contrary to previous reports, Aa-nat transcripts are present not only in the photoreceptor cells, but also in the inner nuclear layer and in the ganglion cell layer. However, the rhythmic expression of Aa-nat mRNA was observed only in photoreceptor cells.

Several studies have demonstrated that norepinephrine (NE) is the critical neurotransmitter for the regulation of gene expression in the pineal gland. We studied the acute effect of NE stimulation in cultured rat pineal glands using Affymetrix rat genome microarray GeneChip probe arrays. Our data demonstrate that NE stimulation affects regulation of several genes; 44 and 29 genes were up- or down-regulated more than 2.5-fold, respectively. As shown in previous studies, arylalkylamine N-acetyltransferase, cyclic AMP responsive element modulator, jun-B and c-fos mRNA levels were increased by NE stimulation. Genes that were not previously reported and increased by NE stimulation in the pineal gland were protein tyrosine phosphatase, nuclear receptors, and activity and neurotransmitter-induced early genes. Unlike up-regulated genes, most of the down-regulated genes were not reported previously. Genes encoding enzymes involved in metabolism and structural proteins were decreased following NE stimulation. Identification of genes affected by NE stimulation would provide valuable information to understanding pineal biology fully.

The suprachiasmatic nucleus (SCN) contains a circadian clock and regulates melatonin synthesis in the pineal gland. Light exposure during the subjective night acutely increases the mRNA levels of the Period (Per)1 gene in the SCN and acutely suppresses melatonin levels in the pineal gland. Activation of N -methyl- d -aspartate (NMDA) receptors in the SCN has been demonstrated to phase-shift the circadian clock in a manner similar to light. We tested the hypothesis that activation of excitatory amino acid (EAA) receptors in the SCN mediates the acute effects of light on Per1 mRNA levels and pineal melatonin. NMDA, injected into the SCN of Syrian hamsters during the night, acutely suppressed melatonin levels in the pineal gland. Both the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5) and the α-amino-3-hydroxy-5-methylisoxazoleproprionic acid (AMPA)/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) inhibited the light-induced increase of Per1 mRNA levels in the SCN. In the same animals, however, these antagonists had no effect on the ability of light to suppress pineal melatonin. These results support the hypothesis that EAA receptor activation in the SCN is necessary for the acute effects of light on Per1 mRNA levels. They also indicate that NMDA receptor activation in the SCN is sufficient but may not be necessary for the acute effects of light on pineal melatonin. These data suggest that there may be at least two different neurochemical cascades that transduce the effects of light in the SCN

Several studies have established that melatonin synthesis occurs in the retina of vertebrates, including mammals. In mammals, a subpopulation of photoreceptors (probably the cones) synthesize melatonin. Melatonin synthesis in the retina is elevated at night and reduced during the day in a fashion similar to events in the pineal gland. Both the MT1 and MT2 melatonin receptors are present in the retina and retinal melatonin does not contribute to circulating levels, suggesting that retinal melatonin acts locally as a neurohormone and/or neuromodulator. Melatonin synthesis in the retina of mammals is under the control of a circadian oscillator, and circadian rhythms in melatonin synthesis and/or release have been described for several species of mammals. These rhythms are present in vivo, persist in vitro, are entrained by light and are temperature compensated. The cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the key enzyme in the melatonin biosynthetic pathway) has allowed studies of the molecular mechanisms responsible for the generation of retinal melatonin rhythmicity. The present review focuses on the cellular and molecular mechanisms that regulate melatonin synthesis. In particular, we discuss how the photic environment and the circadian clock interact in determining melatonin levels, in addition to the role that melatonin plays in retinal physiology.

Many physiological, cellular, and biochemical parameters in the retina of vertebrates show daily rhythms that, in many cases, also persist under constant conditions. This demonstrates that they are driven by a circadian pacemaker. The presence of an autonomous circadian clock in the retina of vertebrates was first demonstrated in Xenopus laevis and then, several years later, in mammals. In X. laevis and in chicken, the retinal circadian pacemaker has been localized in the photoreceptor layer, whereas in mammals, such information is not yet available. Recent advances in molecular techniques have led to the identification of a group of genes that are believed to constitute the molecular core of the circadian clock. These genes are expressed in the retina, although with a slightly different 24-h profile from that observed in the central circadian pacemaker. This result suggests that some difference (at the molecular level) may exist between the retinal clock and the clock located in the suprachiasmatic nuclei of hypothalamus. The present review will focus on the current knowledge of the retinal rhythmicity and the mechanisms responsible for its control.

Several investigations have shown that illumination at night reduces melatonin level in the mammalian pineal, but the effect of night illumination on the retina is not known. In this study retinas were cultured in a flow-through apparatus and then were exposed to light at ZT 18. Light exposure reduced melatonin levels to the daytime level within 30 min. The reduction of melatonin levels was due to a rapid decrease in the activity of the enzyme AA-NAT; AA-NAT mRNA levels were not affected by illumination. Pre-incubation with lactacystin (25 microM) prevented light-induced reduction of AA-NAT activity and melatonin levels. These results demonstrate that melatonin levels in the mammalian retina are affected by light exposure at night, via proteosomal proteolysis of AA-NAT.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently demonstrated to stimulate melatonin synthesis in the rat pineal gland. Circadian rhythms of melatonin concentration are well known. However, it has not been clarified whether PACAP contents in the pineal gland show circadian rhythm. In this study, we measured PACAP contents in the rat pineal gland throughout the day under 12:12 h light-dark cycle or constant dark conditions. A significant fluctuation was observed in the PACAP content under light-dark conditions but not under constant darkness. On the other hand, the pituitary gland showed no significant variation throughout the day under either conditions. These observations suggest that PACAP may participate in the modulation of melatonin synthesis depending on light conditions in the pineal gland.

Adenylate cyclase-activating polypeptide (PACAP) is synthesized in the retinal ganglion cells which terminate on vasoactive intestinal polypeptide neurons in the suprachiasmatic nucleus (SCN), the location of circadian clock. To examine whether PACAP exhibits daily variations in the rat SCN, we measured endogenous PACAP contents throughout the day under 12:12 h light-dark or constant dark conditions. PACAP level was low during the light periods, high during the dark periods, and was stable under constant dark conditions. In the periventricular nucleus of the hypothalamus and cerebral cortex, PACAP content did not show any significant variation throughout the day. Our findings suggest that PACAP content in the SCN may be changed by lighting conditions. Thus, PACAP-containing neurons may play certain roles in the entrainment of circadian rhythms.

In a previous investigation we have demonstrated that neonatal rat pineal may become photosensitive if removed from the animal at post-natal day 1 and cultured for 7 days. In the present study we demonstrated that Aa-nat mRNA, the key enzyme in melatonin biosynthetic pathway, is affected by illumination in photosensitive pineal, and Aa-nat transcription in photosensitive pineal, but not in adult or non photosensitive pineals, is up-regulated by BMAL1:CLOCK.

Day-night variation of the preprosomatostatin mRNA level in the suprachiasmatic nucleus (SCN), the site of the circadian pacemaker, was determined in rats maintained under light-dark cycles by semiquantitative Northern blot hybridization of micropunched tissues. Preprosomatostatin mRNA abundance in the SCN showed significant variation during a day with a peak level at around light on and a trough at around 4 h after light off. This time course was essentially identical with that previously found in blinded rats, indicating that environmental light did not produce an immediate effect on the preprosomatostatin mRNA level in the SCN. Moreover, this rhythm in the preprosomatostatin mRNA appeared specific to the SCN, since levels in the cortex and anterior hypothalamus did not oscillate even under light-dark conditions.

Daily rhythms are a ubiquitous feature of living systems. Generally, these rhythms are not just passive consequences of cyclic fluctuations in the environment, but instead originate within the organism. In mammals, including humans, the master pacemaker controlling 24 h rhythms is localized in the suprachiasmatic nuclei of the hypothalamus. This pacemaker, or circadian clock, has been shown to be responsible for the temporal organization of a wide variety of functions, ranging from sleep and food intake, to physiological measures such as body temperature, heart rate, and hormone release.

The daily light:dark cycle synchronizes the circadian timing system by resetting the phase of the circadian pacemaker on a daily basis. Light acutely increases mRNA levels of the clock genes Per1 and Per2 in the suprachiasmatic nucleus (SCN), the site of the primary circadian pacemaker in mammals. Light is conveyed to the SCN through the retinohypothalamic tract (RHT), an efferent projection from retinal ganglion cells that releases the excitatory amino acid (EAA) neurotransmitter glutamate in the SCN. EAA receptor activation in the SCN is critical for the ability of light to phase-shift the circadian pacemaker. In a previous study, we demonstrated that EAA receptor activation is necessary and sufficient for light to acutely increase Per1 mRNA levels in the SCN. In the current study, we determined whether EAA receptor activation in the SCN is necessary for the ability of light to increase Per2 mRNA levels in the SCN in Syrian hamsters. The NMDA receptor antagonist AP5 and the AMPA/kainate receptor antagonist DNQX inhibited the ability of light and NMDA to acutely increase Per2 mRNA levels in the SCN. In hamsters injected with DNQX, Per1 and Per2 mRNA levels remained slightly elevated in the ventrolateral SCN, suggesting that AMPA/kainate receptor activation in this region is not critical for the effects of light on the circadian pacemaker.

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a light-entrainable circadian pacemaker. Neurons in the SCN are part of a circuit that conveys light information from retinal efferents to the pineal gland. Light presented during the night acutely increases mRNA levels of the circadian clock genes Per1 and Per2 in the SCN, and acutely suppresses melatonin levels in the pineal gland. The present study investigated whether the ability of light to increase Per1 and Per2 mRNA levels and suppress pineal melatonin levels requires sodium-dependent action potentials in the SCN. Per1 and Per2 mRNA levels in the SCN and pineal melatonin levels were measured in Syrian hamsters injected with tetrodotoxin (TTX) prior to light exposure or injection of N-methyl-d-aspartate (NMDA). TTX inhibited the ability of light to increase Per1 and Per2 mRNA levels and suppress pineal melatonin levels. TTX did not, however, influence the ability of NMDA to increase Per1 and Per2 mRNA levels, though it did inhibit the ability of NMDA to suppress pineal melatonin levels. These results demonstrate that action potentials in the SCN are not necessary for NMDA receptor activation to increase Per1 and Per2 mRNA levels, but are necessary for NMDA receptor activation to decrease pineal melatonin levels. Taken together, these data support the hypothesis that the mechanism through which light information is conveyed to the pacemaker in the SCN is separate from and independent of the mechanism through which light information is conveyed to the SCN cells whose efferents suppress pineal melatonin levels.

Several studies have demonstrated that norepinephrine (NE) is the critical neurotransmitter for the regulation of gene expression in the pineal gland. We studied the acute effect of NE stimulation in cultured rat pineal glands using Affymetrix rat genome microarray GeneChip probe arrays. Our data demonstrate that NE stimulation affects regulation of several genes; 44 and 29 genes were up- or down-regulated more than 2.5-fold, respectively. As shown in previous studies, arylalkylamine N-acetyltransferase, cyclic AMP responsive element modulator, jun-B and c-fos mRNA levels were increased by NE stimulation. Genes that were not previously reported and increased by NE stimulation in the pineal gland were protein tyrosine phosphatase, nuclear receptors, and activity and neurotransmitter-induced early genes. Unlike up-regulated genes, most of the down-regulated genes were not reported previously. Genes encoding enzymes involved in metabolism and structural proteins were decreased following NE stimulation. Identification of genes affected by NE stimulation would provide valuable information to understanding pineal biology fully.

Most of the organisms living on earth show 24 hour (circadian) rhythms that are endogenously controlled by biological clocks. In mammals, these rhythms are generated by the circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. However, recent studies have demonstrated that circadian oscillators can be found in many organs and tissues, and it appears that the circadian oscillators in the periphery are not self-sustained, since, in vitro, the oscillation disappears after a few cycles. Although analysis of the clockwork mechanism indicates that the molecular composition of the clock in the SCN and in the peripheral tissues is very similar, the mechanism responsible for the damping of the circadian oscillation in the periphery is unknown. Recent studies have also indicated that the mammalian circadian system is hierarchically organized in that the SCN (i.e., the master circadian pacemaker) controls the peripheral oscillators in order to coordinate the physiological events in an entire body. The mechanisms by which the SCN controls peripheral oscillators are just starting to be elucidated. The aim of this review is to summarize the most recent findings on functioning of these extra-SCN oscillators and the mechanisms the SCN controls peripheral oscillators.

For many years it was believed that daily rhythms in behaviour and physiology were imposed by rhythms in the physical environment to which organisms responded passively. The demonstration that plants and animals do not respond passively to environmental stimuli, but they possess accurate endogenous time-measuring systems (i.e., circadian clocks) engendered renewed interest in the study of biological rhythms. During the last 30 years, thousands of studies have demonstrated that most organisms have an (or more than one) internal circadian clock(s) which oscillates with a period close to the astronomical day (i.e., 24 hours). Currently we are aware of many of the physiological mechanisms underlying the control of circadian rhythmicity, while the improvement of molecular biology techniques has fostered dramatic advancements in our knowledge of the molecular workings of circadian clocks in many different organisms, man included. The present review is an attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks.

The mammalian suprachiasmatic nucleus (SCN) contains the main circadian clock. Neuropeptide Y (NPY) that is released from the intergeniculate leaflet of the lateral geniculate body to the SCN, acts in the SCN to advance circadian phase in the subjective day via the NPY Y2 receptor. We used semi-quantitative in situ hybridization to determine the effect of NPY on circadian clock genes, Period 1 (Per1) and Period 2 (Per2), expression in SCN slices. Addition of NPY to the brain slices in the subjective day resulted in reduction of Per1 and Per2 mRNA levels 0.5 and 2 h after treatment. NPY Y1/Y5 and Y2 agonists decreased Per1 within 0.5 h. These results suggest that NPY may induce phase shifts by mechanisms involving or resulting in reduction of Per1 and Per2 mRNA levels.

Background Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. Results cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. Conclusion The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector.

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals.

Period 1 (Per1), 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) mRNA levels were determined by semi-quantitative in situ hybridization in rat pineal glands. In agreement with previous reports, AA-NAT mRNA levels were rhythmic in light:dark (LD) cycles and the rhythm persisted in constant dim light (DLL). Per1 and Per2 mRNA also showed significant variations both in LD and DLL. AA-NAT and Per1 mRNA levels showed very similar patterns of variations in LD and DLL to one another, whereas Per2 showed a different pattern of expression from AA-NAT and Per1. Exposure to 30 min of light did not affect the expression of the three genes, while exposure to a longer light pulse (1 or 2 h) decreased AA-NAT and Per1 mRNA levels; Per2 mRNA levels were also decreased but only temporarily. Our results demonstrate that Per1 and Per2 expression in the rat pineal is under circadian control, and suggest Per1 may be regulated by the same mechanism which controls the expression of AA-NAT gene. Per2 seem to be controlled by a different mechanism.

Many biochemical, physiological and behavioural processes in organisms ranging from microorganisms to vertebrates exhibit circadian rhythms1. In Drosophila, the gene period (per) is required for the circadian rhythms of locomotor activity and eclosion behaviour2. Oscillation in the levels of per mRNA and Period (dPer) protein in the fly brain is thought to be responsible for the rhythmicity3,4. However, no per homologues in animals other than insects have been identified. Here we identify the human and mouse genes (hPER and mPer, respectively) encoding PAS-domain (PAS, a dimerization domain present in Per, Amt and Sim)-containing polypeptides that are highly homologous to dPer. Besides this structural resemblance, mPer shows autonomous circadian oscillation in its expression in the suprachiasmatic nucleus, which is the primary circadian pacemaker in the mammalian brain5,6. Clock, a mammalian clock gene encoding a PAS-containing polypeptide7,8, has now been cloned: it is likely that the Per homologues dimerize with other molecule(s) such as Clock through PAS–PAS interaction in the circadian clock system.

Daily profiles of somatostatin mRNA expression were investigated in the rat suprachiasmatic nucleus (SCN) by semiquantitative in situ hybridization histochemistry. Under 12 h light/12 h dark conditions, somatostatin mRNA signals were higher during the day time (Zeitgeber time (ZT) 1) than during the night time (ZT 16). This day-night difference was still maintained in constant darkness where the somatostatin mRNA was higher in the subjective day (circadian time (CT) 1) than in the subjective night (CT 16). Together with previous Northern blot hybridization studies, the present observation suggests that the level of somatostatin mRNA in SCN neurons is controlled by the circadian clock, independent of photic environment.

Arginine-vasopressin (AVP) gene expression in the rat suprachiasmatic nucleus (SCN) is subject to daily rhythmic changes. To determine whether this variation is endogenously generated, temporal changes in the SCN AVP mRNA level in constant dark (DD) condition was compared with changes occurring under the light-dark (LD) condition. In both lighting conditions, the presence of a rhythm in AVP mRNA level was observed in the SCN. In LD condition, peak level of AVP mRNA was found during the latter part of the day (zeitgeber time or ZT 8) and trough value during the night at ZT 20. Correspondingly, peak level of AVP mRNA under DD condition was observed during the latter part of the subjective day (circadian time or CT 8) and a trough during the subjective night (CT 20). Under both lighting conditions, a rapid increase and decrease of mRNA around the peak time was also observed. On the other hand, no significant daily variation in AVP mRNA was found in the supraoptic nucleus in both LD and DD conditions. These results provide evidence that a rhythmic change in AVP mRNA level is regulated by a circadian clock intrinsic to the SCN. The phase relationship of AVP mRNA rhythm to peptide rhythm in the SCN is discussed.

Administration of a somatostatin (SS) depletor, cysteamine, markedly reduced SS levels in rat suprachiasmatic nucleus (SCN). At the same time, cysteamine administration induced a circadian rhythm of vasoactive intestinal polypeptide (VIP) content in the SCN, which otherwise remains constant under constant environmental conditions. These results suggest that the stable level of VIP in the SCN under constant conditions is not an intrinsic property of VIP neurons but a consequence of interactions with other components in the SCN.

The suprachiasmatic nucleus (SCN) of the hypothalamus has been established as the locus of the circadian pacemaker in mammals. The SCN is histochemically divided into two subdivisions: dorsomedial and ventrolateral subfields. The dorsomedial SCN is characterized, in part, by dense somatostatin-like immunoreactivity (SS-LI), but its functional significance in circadian pacemaking remains unclear. Our previous study revealed that 24 h SS-LI contents of the SCN in rats kept under light-dark (LD) conditions or blinded by orbital enucleation showed a distinct circadian rhythm. In the present study, 24 h SS-LI contents of the SCN in sighted rats kept under constant darkness (DD) conditions for prolonged periods were measured by enzyme immunoassay. Cellular contents of SS-LI exhibited a clear circadian rhythm on the third day of constant darkness (DD) with a peak at circadian time (CT) 5, corresponding to the time of peak levels found in LD conditions and in enucleated animals. This endogenous free-running rhythm continued to oscillate without attenuation of the amplitude even at 14 days in constant darkness. Moreover, SS-LI rhythm was found to be similar to the vasopressin rhythm in the SCN. In summary, these findings further strengthen the idea that the cellular content of SS-LI in the SCN is under the control of the endogenous circadian pacemaker.

The aim of the present study was to investigate the in vitro expression of Period 1 (Per1), Period 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) genes in the rat pineal gland to understand the mechanism(s) regulating the expression of these genes in this organ. Pineals, when maintained in vitro for 5 days, did not show circadian rhythmicity in the expression of any of the three genes monitored. Norepinephrine (NE) induced AA-NAT and Per1, whereas its effect on Per2 was negligible. Contrary to what was observed in other systems, NE stimulation did not induce circadian expression of Per1. The effect of NE on Per1 level was dose- and receptor subtype-dependent, and both cAMP and cGMP induced Per1. Per1 was not induced by repeated NE - or forskolin - stimulation. Protein synthesis was not necessary for NE-induced Per1, but it was for reduction of Per1 following NE stimulation. Per1 transcription in pinealocytes was activated by BMAL1/CLOCK. Our results indicate that important differences are present in the regulation of these genes in the mammalian pineal. Copyright 2002 S. Karger AG, Basel.