Resetting the brain clock: time course and localization of mPER1 and mPER2 protein expression in suprachiasmatic nuclei during phase shifts

The mechanism whereby brief light pulses reset the mammalian circadian clock involves acute Per gene induction. In a previous study we investigated light-induced expression of mPer1 and mPer2 mRNA in the suprachiasmatic nuclei (SCN), with the aim of understanding the relationship between gene expression and behavioural phase shifts. In the present study, we examine the protein products of mPer1 and mPer2 genes in the core and shell region of SCN for 34 h following a phase-shifting light pulse, in order to further explore the molecular mechanism of photic entrainment. The results indicate that, during the delay zone of the phase response curve, while endogenous levels of mPER1 and mPER2 protein are falling, a light pulse produces an increase in the expression of both proteins. In contrast, during the advance zone of the phase response curve, while levels of endogenous mPER1 and mPER2 proteins are rising, a light pulse results in a further increase in mPER1 but not mPER2 protein. The regional distribution of mPER1 and mPER2 protein in the SCN follows the same pattern as their respective mRNAs, with mPER1 expression in the shell region of SCN correlated with phase advances and mPER2 in the shell region correlated with phase delays.     

Circadian rhythm of Arg–vasopressin contents in the suprachiasmatic nucleus in relation to corticosterone

Circadian rhythms of locomotor activity and adrenal glucocorticoid are controlled by the suprachiasmatic nucleus (SCN), the center of a biological clock, in mammals. Arg–vasopressin (AVP) contents in the SCN play a role in endogenous circadian rhythm during the absence of time cues. The AVP-containing neurons in the SCN are considered to transmit a circadian signal to the other parts of the brain. The circadian rhythms of AVP in the SCN in relation to the plasma corticosterone and locomotor activity were investigated. Under the light–dark cycle, plasma corticosterone levels were reciprocally correlated with the AVP content in the SCN. Under free-running conditions with constant dim light, AVP rhythms were reciprocally synchronized with the locomotor activity. The correlation of AVP with plasma corticosterone is different at different times of the day both under the LD cycle and constant dim light. Dexamethasone (i.p., 0.1 mg/100) increased the AVP contents, and this tendency was significantly greater during the dark period. These results indicate that corticosterone in the blood may regulate the circadian rhythm through AVP variation in the SCN.     

Entrainment and coupling of the hamster suprachiasmatic clock by daily dark pulses

The circadian rhythm of locomotor activity of hamsters kept in constant light (LL) can split into two distinct components that, in steady state, lie 180 degrees apart. The splitting phenomenon is the result of antiphase circadian oscillations between left and right sides of the suprachiasmatic nuclei (SCN), the master circadian clock in mammals. In unsplit hamsters housed in LL, a single dark pulse produces a phase-shift of the wheel-running activity rhythm, accompanied by a transient down-regulation of clock gene expression in the SCN. In the present study, we evaluated the effects of daily 1-hr dark pulses on wheel-running activity rhythm and on the expression of clock and nonclock proteins in the SCN of Syrian hamsters exposed to LL conditions. The results show that a daily 1-hr dark pulse entrained the rhythm of wheel-running activity of unsplit hamsters. In addition, in split animals, unimodal coupling of the two locomotor activity components was produced by daily 1-hr dark pulses. In the SCN, the effects of entrainment and unimodal coupling of the two separate components by dark observed in behavior were also evident in the bilateral expression of the proteins c-FOS, p-ERK, PERIOD 1, and calbindin. These results show that the bilaterally asymmetric SCN clock, underlying split circadian behavior, can be recoupled in phase and entrained by short daily dark exposure, indicating the synchronizing potency of darkness on the main circadian clock.     

Photic Regulation of Map Kinase Phosphatases MKP1/2 and MKP3 in the Hamster Suprachiasmatic Nuclei

MAP kinases (MAPKs) play a key role in photic entrainment signaling in the suprachiasmatic nuclei (SCN), the mammalian circadian clock. The control of MAPKs is a fine balance between specific kinases (MEKs) and phosphatases (MKPs), whose orchestration in the SCN is still unresolved. We have found MKP1/2 and MKP3 immunoreactive-cells in the hamster SCN, whose levels are rapidly increased in response to transient light stimulation in the subjective night (CT 18), when light is able to entrain the clock. Moreover, the expression level of MKP3 varies under light–dark cycles and constant darkness, peaking at noon, when MAPKs are in their activated state and begin their inactivation. These results show a different perspective on MAPKs in the SCN, which includes its regulation by a complex net of phosphatases.     

Day‐length encoding through tonic photic effects in the retinorecipient SCN region

The circadian clock in the suprachiasmatic nucleus (SCN) plays a critical role in seasonal processes by sensing ambient photoperiod. To explore how it measures day‐length, we assessed the state of SCN oscillators using markers for neuronal activity (c‐FOS) and the clock protein (PER1) in Syrian hamsters housed in long (LD, 16 : 8 h light : dark) vs. short days (SD, 8 : 16 h light : dark). During SD, there was no detectable phase dispersion across the rostrocaudal extent of the nucleus. In contrast, during LD, rhythms in the caudal SCN phase led those in the mid‐ and rostral SCN by 4–8 h and 8–12 h, respectively. Importantly, some neurons in the retinorecipient core SCN were unique in that they were FOS‐positive during the dark phase in LD, but not SD. Transfer of LD animals to constant darkness or skeleton photoperiod revealed that dark‐phase FOS expression depends on tonic light exposure rather than on intrinsic clock properties. By transferring animals from SD to LD, we next discovered that there are two separate populations of SCN cells, one responding to acute and the other to tonic light exposure. The results suggest that the seasonal encoding of day‐length by the SCN entails reorganization of its constituent oscillators by a subgroup of neurons in the SCN core that respond to tonic photic cues.     

Cellular levels of messenger ribonucleic acids encoding vasoactive intestinal Peptide and gastrin-releasing Peptide in neurons of the suprachiasmatic nucleus exhibit distinct 24-hour rhythms

There is strong evidence supporting the view that the Suprachiasmatic nucleus (SCN) functions as a circadian clock; however, the neural and molecular events underlying SCN function remain unclear. A specific subpopulation of neurons within the ventrolateral aspect of the SCN that contains three peptides, vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and gastrin-releasing peptide (GRP), play an important role in SCN function. VIP-containing neurons of the SCN receive synapses from photic projections, and co-injection of all three peptides mimics the phase-delaying effects of light on circadian activity rhythms. In principle, the signaling potential of a neuron containing several transmitters may be affected by the concentration ratio of co-released factors; hence, one mechanism by which VIP/PHI/GRP-containing neurons could influence SCN function is by changing the concentration ratio of these peptides throughout the light-dark cycle. The present study was performed to examine this possibility. Relative cellular levels of mRNA encoding both VIP/PHI and GRP were determined within the SCN every 4 h in rats housed in a 14 h light: 10 h dark cycle. Quantitative in situ hybridization revealed a statistically significant (P<0.005) 24-h profile of changes in VIP/PHI mRNA that peaked during the dark phase, and a significant (P<0.005) 24-h profile of changes in GRP mRNA that peaked during the light phase. These data support the interpretation that cellular levels of mRNAs encoding VIP/PHI and GRP within the SCN exhibit distinct profiles of changes throughout the light-dark cycle. Further, these findings are consistent with the hypothesis that the concentration ratio of VIP and PHI to GRP changes over the light-dark cycle, and that this may be an important mechanism by which circadian rhythms are generated or entrained.     

Short-term constant light potentiation of large-magnitude circadian phase shifts induced by 8-OH-DPAT: effects on serotonin receptors and gene expression in the hamster suprachiasmatic nucleus

Nonphotic phase-shifting of mammalian circadian rhythms is thought to be mediated in part by serotonin (5-HT) acting in the suprachiasmatic nucleus (SCN) circadian clock. Previously we showed that brief (1-3 days) exposure to constant light (LL) greatly potentiates nonphotic phase-shifting induced by the 5-HT agonist, (+/-)2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT). Here we investigated potential mechanisms for this action of LL, including 5-HT receptor upregulation and SCN clock gene and neuropeptide gene expression. Autoradiographic analysis of ritanserin inhibition of [3H]8-OH-DPAT binding indicated that LL (approximately 2 days) did not affect 5-HT7 receptor binding in the SCN or dorsal raphe. Measurement of 5-HT1A autoreceptors in the median raphe and 5-HT1B receptors in the SCN also showed no effect of LL. In experiment 2, hamsters held under a 14-h light : 10-h dark photocycle (LD) or exposed to LL for approximately 2 days received an intraperitoneal injection of 8-OH-DPAT or vehicle at zeitgeber time (ZT) 6 or 0 and were killed after 2 h of dark exposure. 8-OH-DPAT suppressed SCN Per1 and Per2 mRNAs at both ZTs, as assessed by in situ hybridization. Per1 mRNA was also suppressed by LL alone. In addition, in situ hybridization of arginine vasopressin (AVP) mRNA and vasoactive intestinal polypeptide mRNA showed that LL significantly suppressed the former but not the latter. The LL-induced suppression of SCN Per1 mRNA and AVP mRNA may be involved in LL-induced potentiation of pacemaker resetting, especially as these data provide additional evidence that LL suppresses circadian pacemaker amplitude, thus rendering the clock more susceptible to phase-shifting stimuli.     

Mitogen‐ and stress‐activated protein kinase 1 modulates photic entrainment of the suprachiasmatic circadian clock

The master circadian clock in mammals, the suprachiasmatic nucleus (SCN), is under the entraining influence of the external light cycle. At a mechanistic level, intracellular signaling via the p42/44 mitogen‐activated protein kinase pathway appears to play a central role in light‐evoked clock entrainment; however, the precise downstream mechanisms by which this pathway influences clock timing are not known. Within this context, we have previously reported that light stimulates activation of the mitogen‐activated protein kinase effector mitogen‐stress‐activated kinase 1 (MSK1) in the SCN. In this study, we utilised MSK1^?/? mice to further investigate the potential role of MSK1 in circadian clock timing and entrainment. Locomotor activity analysis revealed that MSK1 null mice entrained to a 12 h light/dark cycle and exhibited circadian free‐running rhythms in constant darkness. Interestingly, the free‐running period in MSK1 null mice was significantly longer than in wild‐type control animals, and MSK1 null mice exhibited a significantly greater variance in activity onset. Further, MSK1 null mice exhibited a significant reduction in the phase‐delaying response to an early night light pulse (100 lux, 15 min), and, using an 8 h phase‐advancing ‘jet‐lag’ experimental paradigm, MSK1 knockout animals exhibited a significantly delayed rate of re‐entrainment. At the molecular level, early night light‐evoked cAMP response element‐binding protein (CREB) phosphorylation, histone phosphorylation and Period1 gene expression were markedly attenuated in MSK1^?/? animals relative to wild‐type mice. Together, these data provide key new insights into the molecular mechanisms by which MSK1 affects the SCN clock.     

Expression of Circadian Neuropeptides in the Hypothalamus of Adult Mice is Affected by Postnatal Light Experience

The suprachiasmatic nuclei (SCN) of the hypothalamus are the principal pacemaker in mammals, controlling daily, circadian rhythms in physiology and behaviour. Environmental light during development has long-term effects on circadian behaviour, but it is still unclear what the relevant adaptations within the brain are. In the present study, we examined the manifestation of the circadian rhythm of locomotor activity, and the expression of arginine-vasopressin (AVP) and vasointestinal polypeptide (VIP) in the SCN of adult mice reared under different light environments during the suckling period, and synchronised to light/dark cycles after weaning. We found that animals reared under constant light had higher amplitude and more stable activity rhythms, together with lower levels of VIP- and AVP-immunostaining in the SCN, compared to mice reared under light/dark cycles or constant darkness. Differences in AVP expression were also found in the paraventricular nucleus and the supraoptic nucleus, two brain areas which receive SCN projections. These results indicate that the postnatal light experience may affect clock function and clock output, and suggest implications for the control of hormonal homeostasis and circadian behaviour.     

Altered Rhythm of Adrenal Clock Genes, StAR and Serum Corticosterone in VIP Receptor 2-Deficient Mice

The circadian time-keeping system consists of clocks in the suprachiasmatic nucleus (SCN) and in peripheral organs including an adrenal clock linked to the rhythmic corticosteroid production by regulating steroidogenic acute regulatory protein (StAR). Clock cells contain an autonomous molecular oscillator based on a group of clock genes and their protein products. Mice lacking the VPAC2 receptor display disrupted circadian rhythm of physiology and behaviour, and therefore, we using real-time RT-PCR quantified (1) the mRNAs for the clock genes Per1 and Bmal1 in the adrenal gland and SCN, (2) the adrenal Star mRNA and (3) the serum corticosterone concentration both during a light/dark (L/D) cycle and at constant darkness in wild type (WT) and VPAC2 receptor-deficient mice (VPAC2-KO). We also examined if PER1 and StAR were co-localised in the adrenal steroidogenic cells. Per1 and Bmal1 mRNA showed a 24-h rhythmic expression in the adrenal of WT mice under L/D and dark conditions. During a L/D cycle, the adrenal clock gene rhythm in VPAC2-KO mice was phase-advanced by approximately 6?h compared to WT mice and became arrhythmic in constant darkness. A significant 24-h rhythmic variation in the adrenal Star mRNA expression and circulating corticosterone concentration was similarly phase-advanced during the L/D cycle. The loss of adrenal clock gene rhythm in the VPAC2 receptor knockout mice after transfer into constant darkness was accompanied by disappearance of rhythmicity in Star mRNA expression and serum corticosterone concentration. Double immunohistochemistry showed that the PER1 protein and StAR were co-localised in the same steroidogenic cells. Circulating corticosterone plays a role in the circadian timing system and the misaligned corticosterone rhythm in the VPAC2 receptor knockout mice could be involved in their abnormal rhythms of physiology.     

N-Methyl-D-aspartate microinjected into the suprachiasmatic nucleus mimics the phase-shifting effects of light in the diurnal Nile grass rat (Arvicanthis niloticus)

Mammals exhibit circadian rhythms in behavior generated by the suprachiasmatic nucleus (SCN). Exposure to light synchronizes the circadian clock to the environmental light:dark cycle through the release of glutamate into the SCN. In nocturnal animals such as Syrian hamsters, direct application of NMDA to the SCN results in phase shifts similar to those produced by exposure to light. This study was designed to determine if light phase shifts the circadian pacemaker of diurnal Nile grass rats (Arvicanthis niloticus) housed in constant darkness by acting on NMDA-type glutamate receptors in the suprachiasmatic nucleus (SCN). N-Methyl-D-aspartate (NMDA; 0, 1, 10, 50, and 100 mM) was administered through guide cannulae aimed at the SCN at circadian times when light induces phase shifts. Maximal phase delays were attained with 50 mM NMDA, and maximal phase advances were seen after 100 mM NMDA. A phase-response curve (PRC) for NMDA, determined by administering NMDA at each hour over the circadian cycle, resembled the PRC to light in this species. These data support the hypothesis that NMDA-type glutamate receptors play a critical role in mediating the phase shifting effects of light in diurnal, as well as nocturnal, animals. In addition, these data suggest that diurnal grass rats may be less sensitive to the phase shifting properties of NMDA than nocturnal rodents.     

Expression profiles of PER2 immunoreactivity within the shell and core regions of the rat suprachiasmatic nucleus

The circadian clock cells of the mammalian suprachiasmatic nucleus (SCN) generate oscillations in physiology and behavior that are synchronized (entrained) by the external light/dark (LD) cycle. The mechanisms that mediate the effect of light on the core molecular mechanism of the clock are not well understood, but evidence suggests that the Period2 gene, which encodes a key clock regulator (PER2), might be involved. We assessed the expression of PER2 immunoreactivity in the retinorecipient core and shell compartments of the SCN of rats entrained to cycles of discrete light pulses presented at the early subjective day (dawn) or night (dusk), or housed in constant light. We found that in animals entrained to a 0.5 h:23.5-h LD cycle (light falls near dawn), PER2 expression is rhythmic both in the shell and in the core regions of the SCN and indistinguishable from that seen in the SCN of control rats housed in complete darkness. Similarly, the pattern of PER2 expression in the SCN of rats entrained to a 0.5-h:25.5-h LD cycle (light falls near dusk) resembled that in dark-housed controls. We also found that presentation of a discrete light pulse in the early subjective night did not induce PER2 protein expression in the SCN, even 6 h after photic stimulation. Finally, in constant light-housed, behaviorally arrhythmic rats, PER2 expression in the SCN was low and nonrhythmic. These results show that rhythmic PER2 expression occurs both in the shell and core regions of the rat SCN. Furthermore, they show that the expression of PER2 in the SCN is not regulated by entraining light. Finally, constant light-induced behavioral arrhythmicity is associated with a disruption of rhythmic PER2 expression in the whole SCN. Together, the results are consistent with a proposed role for PER2 in the core mechanism of the circadian clock but argue against an important role for PER2 in the mechanism mediating photic entrainment.     

Shedding light on circadian clock resetting by dark exposure: differential effects between diurnal and nocturnal rodents

The master circadian clock in mammals, located in the suprachiasmatic nuclei (SCN) of the hypothalamus, is entrained by light and behavioural stimulation. In addition, the SCN can be reset by dark pulses in nocturnal rodents under constant light conditions. Here, the shifting effects of a dark pulse on the SCN clock were detailed at both a behavioural and molecular level in a nocturnal rodent (Syrian hamster), and were compared to those of a diurnal rodent (Arvicanthis ansorgei). Four-hour dark pulses led to phase advances in the circadian rhythm of locomotor activity from subjective midday to dusk in hamsters, but from subjective dusk to midnight in Arvicanthis. Moreover, dark pulses had no resetting effect during the middle of the subjective night in hamsters, while such a dead shifting zone occurred during most of the subjective day in Arvicanthis. The behavioural phase advances in both hamsters and Arvicanthis were most often accompanied by marked downregulation of the clock genes Per1 and/or Per2 in the SCN, and also by changes in the transforming growth factor-alpha expression, a neuropeptide that suppresses daytime activity in nocturnal mammals. Despite that both hamsters and Arvicanthis showed dark-induced phase advances at circadian time-12, Per1 gene and its protein PER1 were downregulated in Arvicanthis but not in hamsters. Altogether these results show that dark resetting of the SCN is always associated with downregulation of Per1 and/or Per2 expression, and mostly occurs during resting. Thus, the circadian window of sensitivity to dark differs between nocturnal and diurnal rodents.