Site‐specific effects of gastrin‐releasing peptide in the suprachiasmatic nucleus

The effects of gastrin‐releasing peptide (GRP) on the circadian clock in the suprachiasmatic nucleus (SCN) are dependent on the activation of N‐methyl‐d ‐aspartate (NMDA) receptors in the SCN. In this study, the interaction between GRP, glutamate and serotonin in the regulation of circadian phase in Syrian hamsters was evaluated. Microinjection of GRP into the third ventricle induced c‐fos and p‐ERK expression throughout the SCN. Coadministration of an NMDA antagonist or 8‐hydroxy‐2‐di‐n‐propylamino‐tetralin [a serotonin (5‐HT)_1A,7 agonist, DPAT] with GRP limited c‐fos expression in the SCN to a region dorsal to GRP cell bodies. Similar to the effects of NMDA antagonists, DPAT attenuated GRP‐induced phase shifts in the early night, suggesting that the actions of serotonin on the photic phase shifting mechanism occur downstream from retinorecipient cells. c‐fos and p‐ERK immunoreactivity in the supraoptic (SON) and paraventricular hypothalamic nuclei also increased following ventricular microinjection of GRP. Because of this finding, a second set of experiments was designed to test a potential role for the SON in the regulation of clock function. Syrian hamsters were given microinjections of GRP into the peri‐SON during the early night. GRP‐induced c‐fos activity in the SCN was similar to that following ventricular administration of GRP. GRP or bicuculline (a γ‐aminobutyric acid_A antagonist) administered near the SON during the early night elicited phase delays of circadian activity rhythms. These data suggest that GRP‐induced phase‐resetting is dependent on levels of glutamatergic and serotonergic neurotransmission in the SCN and implicate activity in the SON as a potential regulator of photic signaling in the SCN.     

Light- and clock-dependent regulation of ribosomal S6 kinase activity in the suprachiasmatic nucleus

Recent work has revealed that signalling via the p42/44 mitogen-activated protein kinase (MAPK) pathway couples light to entrainment of the circadian clock located in the suprachiasmatic nucleus (SCN). Given that many effects of the MAPK pathway are mediated by intermediate kinases, it was of interest to identify kinase targets of ERK in the SCN. One potential target is the family of 90-kDa ribosomal S6 kinases (RSKs). In this study, we examined light-induced regulation of RSK-1 in the SCN. Immunohistochemical and Western analysis were used to show that photic stimulation during the early and late night triggered the phosphorylation of RSK-1 at two sites that are targeted by ERK. This increase in the phosphorylation state of RSK-1 corresponded with an approximate fourfold increase in kinase activity. Light exposure during the subjective day did not increase the phosphorylated form of RSK-1, indicating that the capacity of light to stimulate RSK-1 activation is phase-restricted. Double immunofluorescent labelling of SCN tissue revealed the colocalized expression of the activated form of ERK with the phosphorylated form of RSK-1 following a light pulse. In vivo pharmacological inhibition of light-induced MAPK pathway activation blocked RSK-1 phosphorylation, indicating that RSK-1 activity is regulated by the MAPK pathway. PDK-1, a coregulator of RSK-1, is also expressed in the SCN and is likely to contribute to RSK-1 activity. RSK-1 phosphorylation was also rhythmically regulated within a subset of phospho-ERK-expressing cells. Together these results identify RSK-1 as a light- and clock-regulated kinase and raise the possibility that it contributes to entrainment and timing of the circadian pacemaker.     

Photic regulation of the mTOR signaling pathway in the suprachiasmatic circadian clock

Here we analyzed the light-responsiveness of the mammalian target of rapamycin (mTOR) cascade, a key regulator of inducible translation, in the suprachiasmatic nuclei (SCN), the locus of the master circadian clock. Brief light exposure during the subjective night, but not during the subjective day, triggered rapid phosphorylation (a marker of catalytic activity) of the mTOR translation effectors p70 S6K, ribosomal S6 protein (S6) and 4E-BP1. In the absence of photic stimulation, marked S6 and 4E-BP1 phosphorylation was detected, indicating tonic mTOR activity in the SCN. Light stimulated the colocalized activation of p70 S6K and extracellular signal-regulated protein kinase (ERK), and pharmacological disruption of ERK signaling abolished light-induced mTOR activity, revealing that the MAPK cascade is an essential intermediate that couples light to mTOR. Together these data identify a light-responsive mTOR cascade in the SCN, and thus, raise the possibility that inducible translation contributes to the clock entrainment process.     

Non-photic phase shifting of the circadian clock: role of the extracellular signal-responsive kinases I/II/mitogen-activated protein kinase pathway

The master circadian clock, located in the suprachiasmatic nucleus (SCN), is synchronized to the external world primarily through exposure to light. A second class of stimuli based on arousal or activity can also reset the hamster circadian clock in a manner distinct from light. The mechanism underlying these non-photic phase shifts is unknown, although suppression of canonical clock genes and immediate early genes has been implicated. Recently, suppression of one of the mitogen-activated protein kinases (MAPK), namely extracellular signal-responsive kinases I/II (ERK), has been implicated in phase shifts to dark pulses, a stimulus with both photic and non-photic components. We investigated the involvement of the ERK/MAPK pathway in phase shifts in response to 3 h of sleep deprivation initiated at mid-day. About three-quarters of animals subjected to this procedure demonstrated large phase advances of about 3 h. Those that shifted exhibited a significant decrease in phosphorylated ERK (p-ERK) in the SCN. Those animals that were perfused during the sleep deprivation also exhibited immunoreactivity for p-ERK in a distinct portion of the ventrolateral SCN. Finally, injections of U0126 to the SCN to prevent phosphorylation of ERK significantly decreased levels of p-ERK but did not produce phase shifts. These data demonstrate that a purely non-photic manipulation is able to alter the activity of the MAPK pathway in the SCN, with downregulation in the SCN shell and activation in a portion of the SCN core.     

Role of vasoactive intestinal peptide in the light input to the circadian system

The neuropeptide vasoactive intestinal peptide (VIP) is expressed at high levels in a subset of neurons in the ventral region of the suprachiasmatic nucleus (SCN). While VIP is known to be important for the synchronization of the SCN network, the role of VIP in photic regulation of the circadian system has received less attention. In the present study, we found that the light‐evoked increase in electrical activity in vivo was unaltered by the loss of VIP. In the absence of VIP, the ventral SCN still exhibited N‐methyl‐d ‐aspartate‐evoked responses in a brain slice preparation, although the absolute levels of neural activity before and after treatment were significantly reduced. Next, we used calcium imaging techniques to determine if the loss of VIP altered the calcium influx due to retinohypothalamic tract stimulation. The magnitude of the evoked calcium influx was not reduced in the ventral SCN, but did decline in the dorsal SCN regions. We examined the time course of the photic induction of Period1 in the SCN using in situ hybridization in VIP‐mutant mice. We found that the initial induction of Period1 was not reduced by the loss of this signaling peptide. However, the sustained increase in Period1 expression (after 30 min) was significantly reduced. Similar results were found by measuring the light induction of cFOS in the SCN. These findings suggest that VIP is critical for longer‐term changes within the SCN circuit, but does not play a role in the acute light response.     

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.     

Serotonergic enhancement of circadian responses to light: role of the raphe and intergeniculate leaflet

Light serves as the primary stimulus that synchronizes the circadian clock in the suprachiasmatic nucleus (SCN) to the external day/night cycle. Appropriately timed light exposure can reset the phase of the circadian clock. Some serotonergic drugs that bind to the serotonin 1A receptor can enhance phase shifts to light. The mechanism by which this potentiation occurs is not well understood. In this study, we examined where one of these drugs, 8‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl]‐8‐azaspiro[4.5]decane‐7,9‐dione dihydrochloride (BMY7378), might be working in the hamster brain. Systemic (5 mg/kg), intra‐dorsal raphe and intra‐median raphe (both 15.6 nmol in 0.5 μL), but not intra‐SCN (7.8 nmol or 15.6 nmol in 0.5 μL) injections of BMY7378 significantly potentiated phase shifts to light. Potentiation of photic shifts persisted when serotonergic innervation of the SCN was lesioned with infusions of the serotonin neurotoxin 5,7‐dihydroxytryptamine into the SCN. Light‐induced c‐Fos expression in the rostral and caudal intergeniculate leaflet (IGL) was attenuated with systemic BMY7378, suggesting that the IGL may be involved in this response. Both complete IGL lesions and depletion of serotonergic innervation of the IGL prevented systemic BMY7378 from potentiating photic phase shifts. Together, these findings suggest that the mechanism by which BMY7378 enhances photic responses is by changing the activity of the raphe nuclei to influence how the IGL responds to light, which subsequently influences the SCN as one of its downstream targets. Identification of the network that underlies this potentiation could lead to the development of useful therapeutic interventions for treating sleep and circadian disorders.     

Mice with early retinal degeneration show differences in neuropeptide expression in the suprachiasmatic nucleus

Background  

In mammals, the brain clock responsible for generating circadian rhythms is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light entrainment of the clock occurs through intrinsically photosensitive retinal ganglion cells (ipRGCs) whose axons project to the SCN via the retinohypothalamic tract. Although ipRGCs are sufficient for photoentrainment, rod and cone photoreceptors also contribute. Adult CBA/J mice, which exhibit loss of rod and cone photoreceptors during early postnatal development, have greater numbers of ipRGCs compared to CBA/N control mice. A greater number of photosensitive cells might argue for enhanced light responses, however, these mice exhibit attenuated phase shifting behaviors. To reconcile these findings, we looked for potential differences in SCN neurons of CBA/J mice that might underly the altered circadian behaviors. We hypothesized that CBA/J mice have differences in the expression of neuropeptides in the SCN, where ipRGCs synapse. The neuropeptides vasoactive intestinal peptide (VIP) and vasopressin (VP) are expressed by many SCN neurons and play an important role in the generation of circadian rhythms and photic entrainment.     

Differential localization of PER1 and PER2 in the brain master circadian clock

The hypothalamic suprachiasmatic nucleus (SCN), locus of the master circadian clock, bears many neuronal types. At the cellular–molecular level, the clock is comprised of feedback loops involving ‘clock’ genes including Period1 and Period2, and their protein products, PERIOD1 and PERIOD2 (PER1/2). In the canonical model of circadian oscillation, the PER1/2 proteins oscillate together. While their rhythmic expression in the SCN as a whole has been described, the possibility of regional differences remains unknown. To explore these clock proteins in distinct SCN regions, we assessed their expression through the rostro‐caudal extent of the SCN in sagittal sections. We developed an automated method for tracking three fluorophores in digital images of sections triply labeled for PER1, PER2, and gastrin‐releasing peptide (used to locate the core). In the SCN as a whole, neurons expressing high levels of PER2 were concentrated in the rostral, rostrodorsal, and caudal portions of the nucleus, and those expressing high levels of PER1 lay in a broad central area. Within these overall patterns, adjacent cells differed in expression levels of the two proteins. The results demonstrate spatially distinct localization of high PER1 vs. PER2 expression, raising the possibility that their distribution is functionally significant in encoding and communicating temporal information. The findings provoke the question of whether there are fundamental differences in PER1/2 levels among SCN neurons and/or whether topographical differences in protein expression are a product of SCN network organization rather than intrinsic differences among neurons.     

Urokinase‐type plasminogen activator modulates mammalian circadian clock phase regulation in tissue‐type plasminogen activator knockout mice

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue‐type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA^?/?; B6.129S2‐Plat^tm1Mlg/J) mice, and identify urokinase‐type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA^?/? mice entrain to a light‐dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA^?/? mice take significantly longer to entrain than C57BL/6J wild‐type (WT) mice. SCN brain slices from tPA^?/? mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose‐dependency. Pre‐treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor‐1 (PAI‐1), inhibits glutamate‐induced phase delays in tPA^?/? slices. Selective inhibition of uPA with UK122 prevents glutamate‐induced phase resetting in tPA^?/? but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA^?/? slices. Casein‐plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA^?/? SCN. Finally, tPA^?/? SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.     

Circadian regulation of mouse suprachiasmatic nuclei neuronal states shapes responses to orexin

Our knowledge of how circadian and homeostatic brain circuits interact to temporally organize physiology and behavior is limited. Progress has been made with the determination that lateral hypothalamic orexin (OXA) neurons control arousal and appetitive states, while suprachiasmatic nuclei (SCN) neurons function as the master circadian clock. During the day, SCN neurons exhibit heterogeneity in spontaneous resting membrane potential (RMP), with some neurons becoming severely depolarized (hyperexcited) and ceasing to fire action potentials (APs), while other neurons rest at moderate RMP and fire APs. Intriguingly, the day phase is when the SCN clock is most readily influenced by arousal, but it is unclear if and how heterogeneity in the excitability state of SCN neurons shapes their response to arousal signals, such as OXA. In whole‐cell recordings we show that during the day OXA recruits GABA‐GABA_A receptor signaling to suppress the RMP of hyperexcited silent as well as moderately hyperpolarized AP‐firing SCN neurons. In the AP‐firing neurons, OXA hyperpolarized and silenced these SCN cells, while in the hyperexcited silent neurons OXA suppressed the RMP of these cells and evoked either AP‐firing, depolarized low‐amplitude membrane oscillations, or continued silence at a reduced RMP. These results demonstrate how the resting state of SCN neurons determines their response to OXA, and illustrate that the inhibitory action of this neurochemical correlate of arousal can trigger paradoxical AP firing.     

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.     

Ryanodine‐sensitive intracellular Ca2+ channels are involved in the output from the SCN circadian clock

The suprachiasmatic nuclei (SCN) contain the major circadian clock responsible for generation of circadian rhythms in mammals. The time measured by the molecular circadian clock must eventually be translated into a neuronal firing rate pattern to transmit a meaningful signal to other tissues and organs in the animal. Previous observations suggest that circadian modulation of ryanodine receptors (RyR) is a key element of the output pathway from the molecular circadian clock. To directly test this hypothesis, we studied the effects of RyR activation and inhibition on real time expression of PERIOD2::LUCIFERASE, intracellular calcium levels and spontaneous firing frequency in mouse SCN neurons. Furthermore, we determined whether the RyR‐2 mRNA is expressed with a daily variation in SCN neurons. We provide evidence that pharmacological manipulation of RyR in mice SCN neurons alters the free [Ca²⁺]_i in the cytoplasm and the spontaneous firing without affecting the molecular clock mechanism. Our data also show a daily variation in RyR‐2 mRNA from single mouse SCN neurons with highest levels during the day. Together, these results confirm the hypothesis that RyR‐2 is a key element of the circadian clock output from SCN neurons.     

Regional circadian period difference in the suprachiasmatic nucleus of the mammalian circadian center

The suprachiasmatic nucleus (SCN) is the mammalian circadian rhythm center. Individual oscillating neurons have different endogenous circadian periods, but they are usually synchronized by an intercellular coupling mechanism. The differences in the period of each oscillating neuron have been extensively studied; however, the clustering of oscillators with similar periods has not been reported. In the present study, we artificially disrupted the intercellular coupling among oscillating neurons in the SCN and observed regional differences in the periods of the oscillating small‐latticed regions of the SCN using a transgenic rat carrying a luciferase reporter gene driven by regulatory elements from a per2 clock gene (Per2::dluc rat). The analysis divided the SCN into two regions – a region with periods shorter than 24 h (short‐period region, SPR) and another with periods longer than 24 h (long‐period region, LPR). The SPR was located in the smaller medial region of the dorsal SCN, whereas the LPR occupied the remaining larger region. We also found that slices containing the medial region of the SCN generated shorter circadian periods than slices that contained the lateral region of the SCN. Interestingly, the SPR corresponded well with the region where the SCN phase wave is generated. We numerically simulated the relationship between the SPR and a large LPR. A mathematical model of the SCN based on our findings faithfully reproduced the kinetics of the oscillators in the SCN in synchronized conditions, assuming the existence of clustered short‐period oscillators.     

Daily and seasonal adaptation of the circadian clock requires plasticity of the SCN neuronal network

Circadian rhythms are an essential property of many living organisms, and arise from an internal pacemaker, or clock. In mammals, this clock resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, and generates an intrinsic circadian rhythm that is transmitted to other parts of the CNS. We will review the evidence that basic adaptive functions of the circadian system rely on functional plasticity in the neuronal network organization, and involve a change in phase relation among oscillatory neurons. We will illustrate this for: (i) photic entrainment of the circadian clock to the light-dark cycle; and (ii) seasonal adaptation of the clock to changes in day length. Molecular studies have shown plasticity in the phase relation between the ventral and dorsal SCN during adjustment to a shifted environmental cycle. Seasonal adaptation relies predominantly on plasticity in the phase relation between the rostral and caudal SCN. Electrical activity is integrated in the SCN, and appears to reflect the sum of the differently phased molecular expression patterns. While both photic entrainment and seasonal adaptation arise from a redistribution of SCN oscillatory activity patterns, different neuronal coupling mechanisms are employed, which are reviewed in the present paper.