Stimuli which entrain the circadian clock of the neonatal Syrian hamster in vivo regulate the phosphorylation of the transcription factor CREB in the suprachiasmatic nucleus in vitro

Photic resetting of the adult mammalian circadian clock in vivo is associated with phosphorylation of the Ser¹³³ residue of the calcium/cyclic AMP response-element binding-protein (CREB) in the retinorecipient region of the suprachiasmatic nucleus (SCN). Western blotting and immunocytochemistry were used to investigate whether agonists known to reset the clock of neonatal hamsters in vivo are also able to influence the phosphorylation of CREB in the suprachiasmatic hypothalamus in vitro. Antisera raised against synthetic CREB peptide sequences were used to differentiate between total CREB and the Ser¹³³ phosphorylated form of CREB (pCREB). Western blot analysis of proteins isolated from suprachiasmatic tissue of 1-day-old Syrian hamsters revealed bands at ≈ 45 kDa corresponding to total CREB and pCREB. Treatment of the tissue with a mixture of glutamatergic agonists [N-methyl-d -aspartate (NMDA), amino-methyl proprionic acid (AMPA) and kainate, all at 1 μm ], or native glutamate (1 μm ) had no effect on the total CREB signal, but increased the pCREB signal, indicative of agonist-stimulated phosphorylation of CREB on Ser¹³³. A similar effect was seen following treatment of the suprachiasmatic blocks with either dopamine (1 μm ) or forskolin (1 μm ). Simultaneous treatment with melatonin (1 μm ) significantly attenuated stimulation by forskolin. The effect of the agonists on nuclear pCREB-immunoreactivity (-ir) was investigated in primary cultures which contained a mixture of cell types characteristic of the suprachiasmatic nuclei in vivo. Basal expression of nuclear total CREB-ir was high, whereas expression of pCREB-ir was low. Treatment with glutamate (1 μm ) or dopamine (1 μm ) had no effect on total CREB-ir, but increased pCREB-ir in ≈ 50 and 30% of cells, respectively, whereas forskolin (1 μm ) increased pCREB-ir in almost all cells (> 90%). The effects of all three agonists were rapid (< 15 min), and dose and time dependent. Melatonin reversed the effects of forskolin in mixed cultures, but not in pure astrocyte cultures. Dual-immunocytochemistry (ICC) revealed that glutamate (1 μm ) increased nuclear pCREB-ir in cells immunoreactive for microtubule-associated protein II (MAP II-ir), but not other cells, indicating an effect predominantly on neurons. This occurred equally in γ-amino butyric acid (GABA)-ir and non-GABA-ir neurons. Dopamine (1 μm ) was more selective, increasing pCREB-ir only in GABA-ir neurons, whereas forskolin increased pCREB-ir in all cells. The specific stimulation of pCREB-ir in GABA-ir neurons by dopamine was reversed by melatonin, but melatonin had no effect on the increase in pCREB-ir induced in GABA-ir neurons by glutamate. These results demonstrate that agonists known to entrain the circadian clock in vivo modulate phosphorylation of CREB in GABA-ir neurons derived from the neonatal suprachiasmatic nuclei.     

Clock gene expressions in the suprachiasmatic nucleus and other areas of the brain during rhythm splitting in CS mice

The CS mouse is a mutant strain which displays spontaneous splitting in the circadian locomotor rhythm under continuous darkness. To clarify whether the rhythm splitting occurs in the suprachiasmatic nucleus (SCN) where the mammalian circadian clock is located, the circadian rhythmicities of mammalian clock genes, mPer1, mBMAL1 and mClock, were examined in the SCN and cerebral cortex during rhythm splitting. The circadian profiles of the clock genes during rhythm splitting were essentially the same as those observed under unsplit conditions. However, the mPer1 gene expression throughout the day was bimodal in the piriform and cingulate cortices, peaking in correspondence with two split components of behavioral rhythm. These results indicate that the circadian profiles of three clock gene expressions in the SCN are not consistent with the overt circadian locomotor rhythm, suggesting that the site of rhythm splitting is somewhere outside the SCN, or alternatively different subregions or other clock genes in the SCN are involved in rhythm splitting.     

Expression of the Per1 gene in the hamster: brain atlas and circadian characteristics in the suprachiasmatic nucleus

Recent progress in study on the molecular component of mammalian clocks has claimed that mammals and Drosophila share the similar fundamental clock oscillating system. In the present study, we investigated expression of Per1, the first gene of the mammalian homolog of the Drosophila clock gene period, in the hamster brain, and we also examined its circadian expression pattern in the mammalian clock center, the suprachiasmatic nucleus (SCN). In situ hybridization using isotope-labeled cRNA probes revealed a wide and region-specific distribution of Per1 in the hamster brain and spinal cord. High levels of Per1 were found in the internal granular layer of the granular cells of the olfactory bulb, anterior olfactory nuclei, tenia tecta, olfactory tubercle, piriform cortex, suprachiasmatic nucleus, and gyrus dentatus of hippocampus. Moderate levels of expression were detected in many brain regions including the granular layer of the cerebellum, anterior paraventricular thalamic nucleus, caudate-putamen, inferior colliculus, pontine nuclei, inferior olive, and nucleus of the solitary tract. We examined the circadian profile of hamster Per1 mRNA in the SCN in constant darkness and found that Per1 expression showed a peak at subjective day (circadian time [CT] 4) and formed a trough at subjective night (CT16-CT20). A brief exposure of light at CT16 could acutely induce large quantities of Per1 mRNA in the hamster SCN, except for its dorsomedial subdivision. These findings suggest that the characteristics of Per1 gene expression in the mammalian circadian center (showing a peak in the daytime and a trough in the nighttime and a rapid inducibility by light) are common among mammalian species. Lastly, in hamster brain, Per1 gene is also inducible in extra-SCN brain nuclei, since light at night also elicited Per1 mRNA in neurons of the hypothalamic paraventricular nucleus.     

Melatonin directly resets the rat suprachiasmatic circadian clock in vitro.

The environmental photoperiod regulates the synthesis of melatonin by the pineal gland, which in turn induces daily and seasonal adjustments in behavioral and physiological state. The mechanisms by which melatonin mediates these effects are not known, but accumulating data suggest that melatonin modulates a circadian biological clock, either directly or indirectly via neural inputs. The hypothesis that melatonin acts directly at the level of the suprachiasmatic nucleus (SCN), a central mammalian circadian pacemaker, was tested in a rat brain slice preparation maintained in vitro for 2-3 days. Exposure of the SCN to melatonin for 1 h late in the subjective day or early subjective night induced a significant advance in the SCN electrical activity rhythm; at other times melatonin was without apparent effect. These results demonstrate that melatonin can directly reset this circadian clock during the period surrounding the day-night transition.     

Two circadian oscillatory mechanisms in the mammalian retina

To investigate the mechanism that controls circadian rhythms in the mammalian retina, we examined the mRNA expression rhythms of serotonin N-acetyltransferase (NAT), the mammalian clock gene rPer2 and a clock-controlled gene Dbp in the retina of rats with lesions of the suprachiasmatic nucleus (SCN), the master clock in mammals. Northern blot analyses showed that retinal NAT mRNA still exhibited the circadian expression in the SCN-lesioned rats, whereas the lesion abolished the rhythms of rPer2 and Dbp mRNAs. These findings suggest that the mammalian retina has two circadian oscillatory mechanisms: one can generate rhythmicity independent of the SCN and the other requires the SCN to maintain circadian oscillation.     

Diurnal regulation of a DNA binding protein to the period repeat sequence in the SCN nuclear extract of rat brain

We previously reported the mammalian period repeat mRNA fluctuates during circadian time in the rat suprachiasmatic nucleus (SCN) which is considered to be a clock pacemaker in mammalian brain. Presently we discovered a period repeat sequence (PR) DNA-binding protein in the rat SCN nuclear extract. In the SCN, the binding activity of PR DNA-binding protein to (ACAGGC)3 was most highest during the late day and most lowest during the late night by electro-mobility shift assay (EMSA). In the cortex nuclear extract, the binding of PR DNA-binding protein did not show a significant variation during a day. This is the first report to show the existence of diurnal regulated PR DNA-binding protein in the SCN.     

Serotonin phase-shifts the mouse suprachiasmatic circadian clock in vitro

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) receives multiple afferent signals that could potentially modulate its phase. One input, the serotonin (5-HT) projection from the raphe nuclei, has been extensively investigated in rats and hamsters, yet its role(s) in modulating circadian clock phase remains controversial. To expand our investigation of 5-HT modulation of the SCN clock, we investigated the phase-shifting effects of 5-HT and its agonist, (+)8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), when applied to mouse SCN brain slices. 5-HT induced 2-3 h phase advances when applied during subjective day, while non-significant phase shifts were seen after 5-HT application at other times. These phase shifts were completely blocked by the 5-HT antagonist, metergoline. DPAT also induced phase shifts when applied during mid-subjective day, and this effect appeared dose-dependent. Together, these results demonstrate that the mouse SCN, like that of the rat, is directly sensitive to in vitro phase-resetting by 5-HT.     

Is light-regulated AP-1 binding in the rat suprachiasmatic nucleus gated by the circadian clock?

In mammals, photic entrainment of circadian rhythms likely involves light- and clock-dependent expression of immediate early genes, including fos-like and jun-like genes, in the rat suprachiasmatic nucleus. Using an electrophoretic mobility shift assay, we evaluated whether the photic regulation of DNA-binding activity and composition of activating protein-1 (AP-1) complexes in the suprachiasmatic nucleus is also dependent on circadian phase. Phase-dependent light inducibility in the expression of fra-2 and c-fos genes and in immunoreactive Fra-2 and c-Fos protein expression was also evaluated, by in situ hybridization and immunocytochemistry. Light's effects on AP-1 DNA-binding differed both qualitatively and quantitatively according to the circadian phase at which light was applied. This phase dependence accounted for by both compartmentalization of proteins involved in constitutive AP-1 complexes within the nucleus or cytoplasm and control of the extent to which the expression of specific complexes was induced. It was then shown that the mechanisms by which the circadian clock gates the photic induction of AP-1 components differed according to the nature of the protein.     

Challenging the omnipotence of the suprachiasmatic timekeeper: are circadian oscillators present throughout the mammalian brain?

The suprachiasmatic nucleus of the hypothalamus (SCN) is the master circadian pacemaker or clock in the mammalian brain. Canonical theory holds that the output from this single, dominant clock is responsible for driving most daily rhythms in physiology and behaviour. However, important recent findings challenge this uniclock model and reveal clock-like activities in many neural and non-neural tissues. Thus, in addition to the SCN, a number of areas of the mammalian brain including the olfactory bulb, amygdala, lateral habenula and a variety of nuclei in the hypothalamus, express circadian rhythms in core clock gene expression, hormone output and electrical activity. This review examines the evidence for extra-SCN circadian oscillators in the mammalian brain and highlights some of the essential properties and key differences between brain oscillators. The demonstration of neural pacemakers outside the SCN has wide-ranging implications for models of the circadian system at a whole-organism level.     

Polysialic acid enters the cell nucleus attached to a fragment of the neural cell adhesion molecule NCAM to regulate the circadian rhythm in mouse brain

In the mammalian nervous system, the neural cell adhesion molecule NCAM is the major carrier of the glycan polymer polysialic acid (PSA) which confers important functions to NCAM's protein backbone. PSA attached to NCAM contributes not only to cell migration, neuritogenesis, synaptic plasticity, and behavior, but also to regulation of the circadian rhythm by yet unknown molecular mechanisms. Here, we show that a PSA-carrying transmembrane NCAM fragment enters the nucleus after stimulation of cultured neurons with surrogate NCAM ligands, a phenomenon that depends on the circadian rhythm. Enhanced nuclear import of the PSA-carrying NCAM fragment is associated with altered expression of clock-related genes, as shown by analysis of cultured neuronal cells deprived of PSA by specific enzymatic removal. In vivo, levels of nuclear PSA in different mouse brain regions depend on the circadian rhythm and clock-related gene expression in suprachiasmatic nucleus and cerebellum is affected by the presence of PSA-carrying NCAM in the cell nucleus. Our conceptually novel observations reveal that PSA attached to a transmembrane proteolytic NCAM fragment containing part of the extracellular domain enters the cell nucleus, where PSA-carrying NCAM contributes to the regulation of clock-related gene expression and of the circadian rhythm.     

Transforming growth factor-alpha is expressed in astrocytes of the suprachiasmatic nucleus in hamster: role of glial cells in circadian clocks

Transforming growth factor-alpha (TGF-alpha) is abundantly expressed in the suprachiasmatic nucleus of several rodent species. It was recently suggested to be a clock output signal regulating the activity/rest rhythm. In this study we further characterized the cellular identity of TGF-alpha-expressing cells in the suprachiasmatic nucleus of the Syrian hamster (Mesocricetus auratus). Using confocal laser scanning fluorescence imaging on brain sections immuno-histologically processed for TGF-alpha and GFAP double staining, we observed that in the suprachiasmatic nucleus, TGF-alpha staining is located mainly in GFAP-positive cells, indicating suprachiasmatic nucleus astrocytes produce TGF-alpha. Glial expression of TGF-alpha was also observed in the 3rd ventricle tanycytes of the retrochiasmatic area. In other brain regions where the TGF-alpha message is abundant, such as in the piriform cortex, we observed that TGF-alpha staining is mainly located in neurons. Our results provide the first evidence that glial cells are involved in the regulation of output from the suprachiasmatic nucleus circadian clock through a diffusible mechanism.     

Localization of cholinergic neurons in the forebrain and brainstem that project to the suprachiasmatic nucleus of the hypothalamus in rat

In mammals, the suprachiasmatic nucleus is responsible for the generation of most circadian rhythms and their entrainment to environmental cues. Cholinergic agents can alter circadian rhythm phase, and fibres immunoreactive for choline acetyltransferase, the biosynthetic enzyme for acetylcholine, are present in the suprachiasmatic nucleus. Since there are no cholinergic somata in the suprachiasmatic nucleus, these fibres must represent the terminals of cholinergic neurons whose cell bodies are located elsewhere in the brain. This study was aimed at locating the cholinergic neurons that project to the suprachiasmatic nucleus by retrograde and anterograde tract-tracing and immunohistochemistry for choline acetyltransferase in the rat. After injection of fluorogold, a retrograde tracer, into the suprachiasmatic nucleus, retrogradely labelled neurons that were immunopositive for choline acetyltransferase were located throughout the rostrocaudal extent of the cholinergic basal nuclear complex, with highest densities in the substantia innominata and the nucleus basalis magnocellularis. A few cells were also located in the medial septum and in the vertical and horizontal limbs ofthe diagonal band of Broca. In the brainstem, double-labelled neurons were located in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus and the parabigeminal nucleus. Injections of the anterograde tracer biocytin in these three brainstem nuclei resulted in fibre labelling in the suprachiasmatic nucleus, consistent with the retrograde findings. No clearly double-labelled cells were located in the retina. These results suggest that the suprachiasmatic nucleus receives cholinergic afferents from both the basal forebrain and mesopontine tegmentum which may mediate cholinergic effects on circadian rhythms. © 1993 Wiley-Liss, Inc.     

The hamster circadian rhythm system includes nuclei of the subcortical visual shell.

The clock regulating mammalian circadian rhythmicity resides in the suprachiasmatic nucleus. The intergeniculate leaflet, a major component of the subcortical visual system, has been shown to be essential for certain aspects of circadian rhythm regulation. We now report that midbrain visual nuclei afferent to the intergeniculate leaflet are also components of the hamster circadian rhythm system. Loss of connections between the intergeniculate leaflet and visual midbrain or neurotoxic lesions of pretectum or deep superior colliculus (but not of the superficial superior colliculus) blocked phase shifts of the circadian activity rhythm in response to a benzodiazepine injection during the subjective day. Such damage did not disturb phase response to a novel wheel stimulus. The amount of wheel running or open field locomotion were equivalent in lesioned and control groups after benzodiazepine treatment. Electrical stimulation of the deep superior colliculus, without its own effect on circadian rhythm phase, greatly attenuated light-induced phase shifts. Such stimulation was associated with increased FOS protein immunoreactivity in the suprachiasmatic nucleus. The results show that the circadian rhythm system includes the visual midbrain and distinguishes between mechanisms necessary for phase response to benzodiazepine and those for phase response to locomotion in a novel wheel. The results also refute the idea that benzodiazepine-induced phase shifts are the consequence of induced locomotion. Finally, the data provide the first indication that the visual midbrain can modulate circadian rhythm response to light. A variety of environmental stimuli may gain access to the circadian clock mechanism through subcortical nuclei projecting to the intergeniculate leaflet and, via the final common path of the geniculohypothalamic tract, from the leaflet to the suprachiasmatic nucleus.