Neonatal Suprachiasmatic Nucleus Research Articles

CHRONO and DEC1/DEC2 compensate for lack of CRY1/CRY2 in expression of coherent circadian rhythm but not in generation of circadian oscillation in the neonatal mouse SCN

Clock genes Cry1 and Cry2, inhibitory components of core molecular feedback loop, are regarded as critical molecules for the circadian rhythm generation in mammals. A double knockout of Cry1 and Cry2 abolishes the circadian behavioral rhythm in adult mice under constant darkness. However, robust circadian rhythms in PER2::LUC expression are detected in the cultured suprachiasmatic nucleus (SCN) of Cry1/Cry2 deficient neonatal mice and restored in adult SCN by co-culture with wild-type neonatal SCN. These findings led us to postulate the compensatory molecule(s) for Cry1/Cry2 deficiency in circadian rhythm generation. We examined the roles of Chrono and Dec1/Dec2 proteins, the suppressors of Per(s) transcription similar to CRY(s). Unexpectedly, knockout of Chrono or Dec1/Dec2 in the Cry1/Cry2 deficient mice did not abolish but decoupled the coherent circadian rhythm into three different periodicities or significantly shortened the circadian period in neonatal SCN. DNA microarray analysis for the SCN of Cry1/Cry2 deficient mice revealed substantial increases in Per(s), Chrono and Dec(s) expression, indicating disinhibition of the transactivation by BMAL1/CLOCK. Here, we conclude that Chrono and Dec1/Dec2 do not compensate for absence of CRY1/CRY2 in the circadian rhythm generation but contribute to the coherent circadian rhythm expression in the neonatal mouse SCN most likely through integration of cellular circadian rhythms.

Roles of Neuropeptides, VIP and AVP, in the Mammalian Central Circadian Clock.

In mammals, the central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Individual SCN cells exhibit intrinsic oscillations, and their circadian period and robustness are different cell by cell in the absence of cellular coupling, indicating that cellular coupling is important for coherent circadian rhythms in the SCN. Several neuropeptides such as arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) are expressed in the SCN, where these neuropeptides function as synchronizers and are important for entrainment to environmental light and for determining the circadian period. These neuropeptides are also related to developmental changes of the circadian system of the SCN. Transcription factors are required for the formation of neuropeptide-related neuronal networks. Although VIP is critical for synchrony of circadian rhythms in the neonatal SCN, it is not required for synchrony in the embryonic SCN. During postnatal development, the clock genes cryptochrome (Cry)1 and Cry2 are involved in the maturation of cellular networks, and AVP is involved in SCN networks. This mini-review focuses on the functional roles of neuropeptides in the SCN based on recent findings in the literature.

Different Roles for VIP Neurons in the Neonatal and Adult Suprachiasmatic Nucleus.

The suprachiasmatic nucleus (SCN) drives circadian rhythms in locomotion through coupled, single-cell oscillations. Global genetic deletion of the neuropeptide Vip or its receptor Vipr2 results in profound deficits in daily synchrony among SCN cells and daily rhythms in locomotor behavior and glucocorticoid secretion. To test whether this phenotype depends on vasoactive intestinal polypeptide (VIP) neurons in the SCN, we ablated VIP SCN neurons in vivo in adult male mice through Caspase3-mediated induction of the apoptotic pathway in cre-expressing VIP neurons. We found that ablation of VIP SCN neurons in adult mice caused a phenotype distinct from Vip- and Vipr2-null mice. Mice lacking VIP neurons retained rhythmic locomotor activity with a shortened circadian period, more variable onsets, and decreased duration of daily activity. Circadian hormonal outputs, specifically corticosterone rhythms, were severely dampened. In contrast, deletion of neonatal SCN VIP neurons dramatically reduced circadian gene expression in the cultured SCN, mimicking the effects of global deletion of Vip or Vipr2. These results suggest that SCN VIP neurons play a role in lengthening circadian period and stimulating the daily surge in glucocorticoids in adults and in synchronizing and sustaining daily rhythms among cells in the developing SCN.

Two coupled circadian oscillations regulate Bmal1-ELuc and Per2-SLR2 expression in the mouse suprachiasmatic nucleus

Circadian rhythms in clock genes, Bmal1 and Per2 expression were monitored simultaneously in the cultured slice of mouse suprachiasmatic nucleus (SCN) by dual bioluminescent reporters. In the neonatal SCN, the phase-relation between the Bmal1 and Per2 rhythms were significantly changed during culture. Medium exchange produced phase-dependent phase shifts (PRCm) in the Bmal1 rhythms, but not in the Per2 rhythms. As a result, the two circadian rhythms were temporally dissociated after medium exchange. In the adult SCN, the phase-relation between the two rhythms was kept constant during culture at least up to 20 cycles. The amplitude of PRCm in the adult SCN was significantly attenuated in the Bmal1 rhythm, whereas a PRCm was developed in the Per2 rhythm. The circadian period was not systematically affected by medium exchange in either of rhythms, regardless of whether it was in the neonatal or the adult SCN. Tetrodotoxin, a sodium channel blocker, enhanced the phase-response in both rhythms but abolished the phase-dependency. In addition, tetrodotoxin lengthened the circadian period independent of the phase of administration. Thus, the Bmal1 and Per2 rhythms in the SCN are dissociable and likely regulated by distinct circadian oscillators. Bmal1 is the component of a Bmal1/REV-ERBa/ROR loop and Per2 a Per/Cry/BMAL1/CLOCK loop. Both loops could be molecular mechanisms of the two circadian oscillators that are coupled through the protein product of Bmal1. The coupling strength between the two oscillations depends on developmental stages.

Casein Kinase 1 Delta (CK1δ) Regulates Period Length of the Mouse Suprachiasmatic Circadian Clock In Vitro

BackgroundCasein kinase 1 delta (CK1δ) plays a more prominent role in the regulation of circadian cycle length than its homologue casein kinase 1 epsilon (CK1ε) in peripheral tissues such as liver and embryonic fibroblasts. Mice lacking CK1δ die shortly after birth, so it has not been possible to assess the impact of loss of CK1δ on behavioral rhythms controlled by the master circadian oscillator in the suprachiasmatic nuclei (SCN).Methodology/Principal FindingsIn the present study, mPER2::LUCIFERASE bioluminescence rhythms were monitored from SCN explants collected from neonatal mice. The data demonstrate that SCN explants from neonatal CK1δ-deficient mice oscillate, but with a longer circadian period than littermate controls. The cycle length of rhythms recorded from neonatal SCN explants of CK1ε-deficient mice did not differ from control explants.Conclusions/SignificanceThe results indicate that CK1δ plays a more prominent role than CK1ε in the maintenance of 24-hour rhythms in the master circadian oscillator.

Neuronal expression of catechol O-methyltransferase mRNA in neonatal rat suprachiasmatic nucleus.

We examined the expression profile of catechol O-methyltransferase (COMT) mRNA and its protein in the neonatal rat hypothalamus by in situ hybridization and immunohistochemistry to clarify the sites of dopamine degradation. Strong COMT mRNA expression was observed in the suprachiasmatic nucleus (SCN) throughout its rostrocaudal extent at postnatal day 1 (P1) and P2, and the mRNA levels decreased gradually until P16. COMT mRNA was predominantly localized to the ventral and medial parts of the SCN. Intense COMT immunoreactivity was demonstrated in the ventral SCN and was detected in neuronal perikarya and processes at P1. Ependymal and microglial cells also exhibited strong COMT immunoreactivity. These results indicate that COMT may directly be involved in dopaminergic signaling in the neonatal SCN.

Developmental changes in calbindin-D28k and calretinin expression in the mouse suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus, the primary circadian pacemaker in mammals, and the retinohypothalamic tract, the retinal afferent fibres to the suprachiasmatic nucleus, both mature during early postnatal life. The establishment of circadian rhythms is thought to depend on input from the retina, but the mechanism remains unknown. Here we examined developmental changes in the expression of the Ca2+-binding proteins calbindin-D28k and calretinin in the mouse hypothalamus. Robust calbindin-D28k immunoreactivity was observed in the dorsomedial suprachiasmatic nucleus and the supraoptic nucleus in neonatal mice (postnatal day 3). The calbindin-D28k immunoreactivity decreased significantly in the suprachiasmatic nucleus but not in the supraoptic nucleus during postnatal days 9-15, when retinohypothalamic tract projections to the suprachiasmatic nucleus are completed. Calretinin immunoreactivity was low in the neonatal suprachiasmatic nucleus and increased with development in the ventrolateral suprachiasmatic nucleus, in parallel with the developmental reduction of calbindin-D28k immunoreactivity observed in the dorsomedial suprachiasmatic nucleus. Developmentally stable calretinin immunoreactivity was also observed in retinohypothalamic tract fibres. Organotypic slice cultures of the suprachiasmatic nucleus were prepared from postnatal day 3 mice to examine the effect of the absence of retinohypothalamic tract inputs on developmental changes in calbindin-D28k and calretinin expression. After 12 days in vitro, the cultured suprachiasmatic nucleus slices exhibited dense calbindin-D28k immunoreactivity similar to neonatal mice, and calretinin immunoreactivity in the ventrolateral suprachiasmatic nucleus similar to young adult mice. These results demonstrate a developmental reduction in calbindin-D28k expression that paralleled retinohypothalamic tract formation and a developmental increase in calretinin expression that is independent of retinohypothalamic tract connections to suprachiasmatic nucleus neurons.

Dopaminergic signalling in the rodent neonatal suprachiasmatic nucleus identifies a role for protein kinase A and mitogen-activated protein kinase in circadian entrainment.

The circadian clock of the suprachiasmatic nuclei (SCN) of perinatal rodents is entrained by maternally derived cues. The SCN of neonatal Syrian hamsters express high-affinity D1 dopamine receptors, and the circadian activity-rest cycle of pups can be entrained by maternal injection of dopaminergic agonists. The present study sought to characterize the intracellular pathways mediating dopaminergic signalling in neonatal rodent SCN. Both dopamine and the D1 agonist SKF81297 caused a dose-dependent increase in phosphorylation of the transcriptional regulator Ca2+/cyclic AMP response element (CRE) binding protein (CREB) in suprachiasmatic GABA-immunoreactive (-IR) neurons held in primary culture. The D1 antagonist SCH23390 blocked this effect. Dopaminergic induction of pCREB-IR in GABA-IR neurons was also blocked by a protein kinase A (PKA) inhibitor, 5-24, and by the MAPK inhibitor, PD98059, whereas KN-62, an inhibitor of Ca2+/calmodulin-dependent (CAM) kinase II/IV was ineffective. Treatment with NMDA increased the level of intracellular Ca2+ in the cultured primary SCN neurons in Mg2+-free medium, but SKF81297 did not. Blockade of CaM kinase II/IV with KN-62 inhibited glutamatergic induction of pCREB-IR in GABA-IR neurons, whereas 5-24 was ineffective, confirming the independent action of Ca2+- and cAMP-mediated inputs on pCREB. SKF81297 caused an increase in pERK-IR in SCN cells, and this was blocked by 5-24, indicative of activation of MAPK via D1/cAMP. These results demonstrate that dopaminergic signalling in the neonatal SCN is mediated via the D1-dependent activation of PKA and MAPK, and that this is independent of the glutamatergic regulation via Ca2+ and CaM kinase II/IV responsible for entrainment to the light/dark cycle.

Expression of estrogen receptor -alpha and -beta immunoreactivity in the cultured neonatal suprachiasmatic nucleus: with special attention to GABAergic neurons.

This study investigated the expression patterns of estrogen receptor -alpha (ERalpha) and -beta (ERbeta) in cultured cells of the suprachiasmatic nucleus (SCN) in neonatal rats by combined application of cell culture and double-label immunocytochemistry. The results revealed that the immunoreactivity for either ERalpha or ERbeta (with predominance of ERbeta) was localized in not only neurons but also astrocytes. The co-expression of both ERalpha and ERbeta in the same individual cell was also demonstrated by the double-label immunocytochemistry. The observations also provide a direct evidence for the differential expression of ER subtypes within GABAergic SCN neurons in vitro and suggest that estrogen's effect on the SCN may be mediated at least in part by its ER-containing GABAergic neurons.

Functional Morphology of the Suprachiasmatic Nucleus

In mammals, the biological clock (circadian oscillator) is situated in the suprachiasmatic nucleus (SCN), a small bilaterally paired structure just above the optic chiasm. Circadian rhythms of sleep–wakefulness and hormone release disappear when the SCN is destroyed, and transplantation of fetal or neonatal SCN into an arrhythic host restores rhythmicity. There are several kinds of peptide-synthesizing neurons in the SCN, with vasoactive intestinal peptide, arginine vasopressin, and somatostatine neurons being most prominent. Those peptides and their mRNA show diurnal rhythmicity and may or may not be affected by light stimuli. Major neuronal inputs from retinal ganglion cells as well as other inputs such as those from the lateral geniculate nucleus and raphe nucleus are very important for entrainment and shift of circadian rhythms. In this review, we describe morphological and functional interactions between neurons and glial elements and their development. We also consider the expression of immediate-early genes in the SCN after light stimulation during subjective night and their role in the mechanism of signal transduction. The reciprocal interaction between the SCN and melatonin, which is synthesized in the pineal body under the influence of polysynaptic inputs from the SCN, is also considered. Finally, morphological and functional characteristics of clock genes, particularly mPers, which are considered to promote circadian rhythm, are reviewed.

Expression of basic helix-loop-helix/pas genes in the mouse suprachiasmatic nucleus

The suprachiasmatic nuclei contain a circadian clock that drives rhythmicity in physiology and behavior. In mice, mutation of the Clock gene produces abnormal circadian behavior [Vitaterna M. H. et al. (1994) Science 264, 715–725]. The Clock gene encodes a protein containing basic helix-loop-helix and PAS (PER-ARNT-SIM) domains [King D. P. et al. (1997) Cell 89, 641–653]. The PAS domain may be an important structural feature of a subset of genes involved in photoreception and circadian rhythmicity. The expression and regulation of messenger RNAs encoding eight members of the basic helix-loop-helix/PAS protein superfamily were examined by in situ hybridization. Six of the genes studied (aryl hydrocarbon receptor nuclear transporter, aryl hydrocarbon receptor nuclear transporter-2, Clock, endothelial PAS-containing protein, hypoxia-inducible factor1α and steroid receptor coactivator-1) were expressed in the suprachiasmatic nucleus of adult and neonatal mice. No evidence for rhythmicity of expression was observed when comparing brains collected early in the subjective day (circadian time 3) with those collected early in subjective night (circadian time 15). Neuronal PAS-containing protein-1 messenger RNA was expressed in the suprachiasmatic nucleus of adult (but not neonatal) mice, and a low-amplitude rhythm of neuronal PAS-containing protein-1 gene expression was detected in the suprachiasmatic nucleus. Neuronal PAS-containing protein-2 messenger RNA was not detected in adult or neonatal suprachiasmatic nucleus. Exposure to light at night (30 or 180 min of light, beginning at circadian time 15) did not alter the expression of any of the genes studied. The expression of multiple members of the basic helix-loop-helix/PAS family in the suprachiasmatic nucleus suggests a rich array of potential interactions relevant to the regulation of the suprachiasmatic circadian clock.

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 approximately 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 microM], or native glutamate (1 microM) 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 microM) or forskolin (1 microM). Simultaneous treatment with melatonin (1 microM) 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 microM) or dopamine (1 microM) had no effect on total CREB-ir, but increased pCREB-ir in approximately 50 and 30% of cells, respectively, whereas forskolin (1 microM) 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 microM) 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 gamma-amino butyric acid (GABA)-ir and non-GABA-ir neurons. Dopamine (1 microM) 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.

Nicotine administration differentially affects gene expression in the maternal and fetal circadian clock

Exposure to nicotine by active and passive cigarette smoke is a common public health problem. Recent studies have demonstrated that human fetuses are also exposed to significant levels of nicotine and that there is a five-fold increase in the incidence of Sudden Infant Death Syndrome among infants born to smoking mothers. We examined the effect of nicotine administration and expression of the immediate early gene c- fos in the maternal and fetal rat brain by in situ hybridization. Nicotine injection (1 mg/kg s.c.) on embryonic day 20 (E20) induced detectable c- fos mRNA in the maternal habenula and hypothalamic paraventricular nucleus whereas, in the fetal brain, c- fos was induced in both these structures and also in the suprachiasmatic nucleus (SCN). Nicotine-induced c- fos expression in the fetal SCN was confirmed by Northern analysis and found to return to near basal levels by 3 h post-injection. These responses were blocked by pre-administration of mecamylamine, indicating that the effect of nicotine is mediated through the cholinergic system. Investigation of the development of this response revealed that nicotine failed to induce c- fos expression in the SCN on E16, caused minimal expression on E18, robust expression on E20 and postnatal day 0 (P0), and no expression on P2 or thereafter. These observations suggest that an alteration in the composition of the nicotinic receptors (nAChR), or the subsequent intracellular responses leading to c- fos expression, occurs in the SCN during the perinatal period. Induction of c- fos mRNA in the SCN by light has been associated with phase-shifts of the circadian system, however, the behavioral consequences of the transient sensitivity of the fetal and neonatal SCN to nicotine administration and the consequences for maternal-fetal entrainment remain to be directly determined.

Transplantation of the suprachiasmatic nucleus in the rat.

Restoration of the circadian rhythmicity in wheel-running activity was shown in rats with bilateral suprachiasmatic nuclei (SCN) lesions, after transplantation of the neonatal SCN into the wall of the third ventricle. Free-running circadian rhythms of the wheel-running activity were recorded in young adult rats at least for a month under constant dark condition. Then, bilateral SCNs were completely lesioned electrolytically under deep pentobarbital anaesthesia. After further recording for more than two months without obvious circadian rhythmicity in wheel-running activity, the animals were subjected to transplantation of the SCN. SCNs taken from day 1 neonatal rats were transplanted by injecting the grafts into the third ventricle of the host rat under pentobarbital anaesthesia. After recovery from the procedure, the rat was returned to a cage with a running wheel. Food and water were available at all times. Successful transplantation led to restoration of the circadian rhythmicity starting from two weeks and up to three months after the transplantation. To identify the SCN in the transplanted graft, we used an immunohistochemical staining method for the VIP (vasoactive intestinal polypeptide) and vasopressin. The VIP was located particularly in the ventral area of the SCN, whereas vasopressin was in the dorsal area. In most cases, where circadian rhythmicity was successfully restored, the graft was attached to the caudal wall of the third ventricle.

Pubertal changes in the medio-basal hypothalamic area after neonatal suprachiasmatic nucleus lesions in the rat.

In order to get more insight into the mechanism by which the onset of puberty is controlled, a developmental study on the displacement of catalase- and dopamine-containing cells in the hypothalamic region was done in rats which received a neonatal lesion of the suprachiasmatic nucleus. The displacement of cells is delayed after these lesions. However, the time lost at the beginning of the displacement is made up at the end of the migration.

Transplantation of the neonatal suprachiasmatic nuclei into rats with complete bilateral suprachiasmatic lesions.

The suprachiasmatic nuclei (SCN) obtained from neonatal day 1 rats were transplanted into the third ventricle of SCN lesioned rats which had shown circadian arrhythmicity in wheel-running activity for more than 2 months. In 8 out of 16 rats that received SCN transplantation, appearance of circadian rhythms in wheel-running activity was observed between 2 and 9 weeks after transplantation. Histological examination revealed ingrowth of the grafts into the periventricular zone, caudal from the lesioned SCN. These findings suggest that the recovery of circadian rhythmicity was the result of functional reinnervation of the periventricular zone by efferent fibers from the SCN.

Neonatal suprachiasmatic nucleus lesions: Effects on the development of circadian rhythms in the rat

Previous studies of the effects of suprachiasmatic nucleus (SCN) destruction and visual pathway transections in adult rodents have revealed the primary significance of the SCN and the retinohypothalamic (RH) projection in the generation and entrainment of circadian rhythms. In the present study we found that complete ablation of the SCN in 2-day-old rats, prior to its innervation by the RH projection, permanently eliminates circadian rhythms in spontaneous locomotor activity and drinking; activity and drinking appear randomly distributed over the light-dark cycle. In addition, females exhibit long periods of constant vaginal cornification and an absence of normal estrous cycles. These effects are independent of the animal's visual status; that is, they occur in blinded as well as sighted animals. Incomplete SCN lesions results in partial disruption of rhythmic functions such as damping of circadian rhythms in activity and/or drinking, irregular estrous cycling, and/or complete disruption of only one or two of these measures of rhythmicity. The absence of spared functions after early SCN destruction is consistent with the high degree of specificity for the SCN exhibited by developing RH fibers and further emphasizes the significance of the SCN in circadian rhythm generation. Neither morphological nor functional plasticity has been found following neonatal ablation of the SCN in the rat.

Neonatal suprachiasmatic nucleus ablation: absence of functional and morphological plasticity.

Neonatal ablation of the suprachiasmatic nucleus in the rat has two important consequences. First, the direct projection from the retina to the suprachiasmatic nucleus fails to develop and no other retinal projection to any hypothalamic nucleus is formed. Second, circadian rhythms in drinking and spontaneous locomotor activity to not appear in these rats when they are tested as adults, and the females exhibit constant vaginal estrus. These observations indicate that the central neural mechanisms responsible for the generation and entrainment of circadian rhythmicity in the rat are not capable of either the functional or morphological plasticity characteristic of other developing neural systems.