c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms.

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.

Transduction of light in the suprachiasmatic nucleus: evidence for two different neurochemical cascades regulating the levels of Per1 mRNA and pineal melatonin.

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 alpha-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

Photic and circadian regulation of retinal melatonin in mammals.

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.

Photic regulation of melatonin in rat retina and the role of proteasomal proteolysis.

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.

Neuropeptide Y rapidly reduces Period 1 and Period 2 mRNA levels in the hamster suprachiasmatic nucleus.

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.

Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse.

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.

Circadian clocks: what makes them tick?

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.

Circadian expression of period 1, period 2, and arylalkylamine N-acetyltransferase mRNA in the rat pineal gland under different light conditions.

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.

Pituitary adenylate cyclase-activating polypeptide rhythm in the rat pineal gland.

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.

The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity.

We describe a hypothalamus-specific mRNA that encodes preprohypocretin, the putative precursor of a pair of peptides that share substantial amino acid identities with the gut hormone secretin. The hypocretin (Hcrt) protein products are restricted to neuronal cell bodies of the dorsal and lateral hypothalamic areas. The fibers of these neurons are widespread throughout the posterior hypothalamus and project to multiple targets in other areas, including brainstem and thalamus. Hcrt immunoreactivity is associated with large granular vesicles at synapses. One of the Hcrt peptides was excitatory when applied to cultured, synaptically coupled hypothalamic neurons, but not hippocampal neurons. These observations suggest that the hypocretins function within the CNS as neurotransmitters.

Circadian oscillation of a mammalian homologue of the Drosophila period gene.

Many biochemical, physiological and behavioural processes in organisms ranging from microorganisms to vertebrates exhibit circadian rhythms. In Drosophila, the gene period (per) is required for the circadian rhythms of locomotor activity and eclosion behaviour. Oscillation in the levels of per mRNA and Period (dPer) protein in the fly brain is thought to be responsible for the rhythmicity. 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 suprachiasmaticnucleus, which is the primary circadian pacemaker in the mammalian brain. Clock, a mammalian clock gene encoding a PAS-containing polypeptide, 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.

Day-night variation of pituitary adenylate cyclase-activating polypeptide (PACAP) level in the rat suprachiasmatic nucleus.

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.

Survival and development of reimplanted fetal epiphyses. An experimental study.

An experiment was undertaken to study the survival and development of fetal epiphyses after excision and reimplantation in rats. The proximal part of fibula in the hindlimb was dissected free from surrounding tissues and then reimplanted again. Of 80 fetuses that were operated on, nine that had been operated on survived to birth. Histological examination on the hindlimbs of these rats at four and six weeks after birth showed that the reimplanted segments survived, and the bony nuclei of the epiphyses were present. Radiographically, at 6 weeks old, secondary centres of ossification at proximal ends of the fibulas could be seen in both operated and normal limbs. These results indicate the clinical possibility of correcting congenital musculoskeletal abnormalities in the future by in utero transplantation of epiphyses.

Differences of somatostatin mRNA in the rat suprachiasmatic nucleus under light-dark and constant dark conditions: an analysis by in situ hybridization.

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.

Phase advances of circadian rhythms in somatostatin depleted rats: effects of cysteamine on rhythms of locomotor activity and electrical discharge of the suprachiasmatic nucleus.

Somatostatin is synthesized in the suprachiasmatic nucleus (SCN), a circadian pacemaker in mammals. To explore the functional significance of somatostatin in the circadian system, we examined rhythms of rat locomotor activity and electrical firing rate of SCN neurons in the brain slice after temporal depletion of somatostatin levels in the SCN. Intraperitoneal administration of cysteamine (200 mg/kg), a somatostatin depletor, significantly reduced somatostatin level in the in vivo SCN 5 min after injection and kept low level as long as 3 to 4 days. This administration, on the other hand, induced significant phase advances of about 51 min in the subsequent free-running rhythm of locomotor activity of the rat. A marked phase advance in the circadian rhythm of firing rate in the SCN was also observed after administration of cysteamine in coronal hypothalamic slices. These persistent phase shifts after administration of a somatostatin depletor may suggest that the change of somatostatin level in the SCN have a feedback influence on the circadian pacemaker.

Circadian variation of arginine-vasopressin messenger RNA in the rat suprachiasmatic nucleus.

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.

Emergence of VIP rhythmicity following somatostatin depletion in the rat suprachiasmatic nucleus.

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.

Day-night variation of preprosomatostatin messenger RNA level in the suprachiasmatic nucleus.

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.

Processing of photic information within the intergeniculate leaflet of the lateral geniculate body: assessed by neuropeptide Y immunoreactivity in the suprachiasmatic nucleus of rats.

Entrainment of the circadian pacemaker in the suprachiasmatic nucleus is accomplished by two neural pathways, the retinohypothalamic and geniculohypothalamic tracts. The geniculohypothalamic tract, which originates from the intergeniculate leaflet and a portion of the ventral lateral geniculate nucleus, is composed of fibers immunoreactive to neuropeptide Y. To assess the processing of photic information by the geniculohypothalamic tract, neuropeptide Y immunoreactivity in the suprachiasmatic nucleus of rats kept under various external lighting conditions was determined by enzyme immunoassay of micropunched tissues. Neuropeptide Y levels in the suprachiasmatic nucleus steadily increased when rats were exposed to continuous light and reached a peak in 2 h before returning to basal level. The amount of increase did not depend on intensity and duration of light exposure. A light pulse as short as 5 min elicited a similar rise in neuropeptide Y, indicating that the response is due to the sudden transition from dark to light. This response, however, was only observed when the dark to light transition occurred at circadian time 0 (subjective dawn) of the pacemaker. A light pulse at circadian time 0, which effectively induces the increase in neuropeptide Y level, does not significantly shift the phase of the circadian rhythm. This observation indicates that the photic pathway utilizing neuropeptide Y may be functional only when the endogenous circadian rhythm is synchronized to external light and dark cycles. Administration of an excitatory amino acid antagonist (MK-801) blocked the increase of neuropeptide Y by light, while an agonist (N-methyl-D-aspartate) induced similar facilitatory effects to that of light on the neuropeptide Y level in the rat suprachiasmatic nucleus. These results suggest that the geniculohypothalamic tract processes photic information so as to facilitate distinction of the transition between light and darkness that occurs either at subjective dawn or dusk.

Substance P-like immunoreactivity in the suprachiasmatic nucleus of the rat.

The content of substance P (SP)-like immunoreactivity (LI) within the suprachiasmatic nucleus (SCN) of rats was determined by enzyme immunoassay to evaluate the effect of light on SP-LI in the rat SCN. Male rats were kept under various lighting conditions: light-dark cycles, constant darkness, continuous light exposure for 24 h or light pulse interrupting constant darkness. Animals were also subjected to ocular enucleation. The present study showed that SP-LI in the SCN was unaffected by environmental lighting conditions or by bilateral ocular enucleation. Immunohistochemical studies also confirmed that SP immunoreactivity, which was found in the ventrolateral (VL) subdivision of the SCN, was not reduced significantly even after ocular enucleation. These results suggest that, in contrast to other neurotransmitters in the VL portion of the SCN such as vasoactive intestinal polypeptide (VIP), gastrin releasing peptide (GRP) and neuropeptide Y (NPY), SP level in the SCN is quite stable to light and arises from an area other than the retina.