Suprachiasmatic Nucleus Neurons Research Articles

Cannabinoid Signaling Recruits Astrocytes to Modulate Presynaptic Function in the Suprachiasmatic Nucleus.

Circadian rhythms are 24-h cycles in physiology regulated by the suprachiasmatic nucleus (SCN) in the brain, where daily cues act on SCN neurons to alter clock timing. Cannabinoid signaling modulates SCN neuronal activity, although the mechanism remains unclear. We propose that neuronal activity generates endocannabinoid release, activating astrocyte Ca2+ signaling, which releases adenosine and activates adenosine-1 receptors (A1Rs) on the presynaptic axon terminals, decreasing GABA release. We demonstrated, in mice, that activation of cannabinoid-1 receptors (CB1R) with the agonist WIN 55,212-2 (WIN) reduced the miniature GABA receptor-mediated postsynaptic current (mGPSC) frequency by a mechanism that requires astrocytes and A1R. WIN activated an intracellular Ca2+ signaling pathway in astrocytes. Activating this intracellular Ca2+ pathway with designer receptors exclusively activated by designer drugs (DREADDs) also decreased the mGPSC frequency and required A1R activation. The frequency of spontaneous Ca2+ events, including those induced by depolarization of a postsynaptic SCN neuron, was reduced by blocking CB1R activation with AM251, demonstrating neuronal endocannabinoid signaling modulates astrocytic Ca2+ signaling in the SCN. Finally, daytime application of WIN or adenosine phase advanced the molecular circadian clock, indicating that this cannabinoid signaling pathway is vital for the timing of circadian rhythms.

Characteristic of Dopamine-Producing System and Dopamine Receptors in the Suprachiasmatic Nucleus in Rats in Ontogenesis.

One of the features of the developing suprachiasmatic nucleus (SCN), the "biological clock" of the body, is the early expression of dopamine (DA) receptors in the absence of dopaminergic neurons as a source of DA. Only recently we showed that DA in SCN is synthesized together by nerve fibers containing only tyrosine hydroxylase (TH) and neurons containing only aromatic L-amino acid decarboxylase (AADC). This study was aimed to assess specific characteristics of the phenotype of TH-fibers in ontogenesis. For this purpose, PCR and immunohistochemical analysis of the expression of genes and proteins such as TH, AADC, vesicular monoamine transporter (VMAT), and receptors for DA (D1, D2) was performed. We have detected numerous TH-immunoreactive fibers in SCN of young and adult rats. VMAT was observed in some of them, which suggests vesicular storage of L-DOPA. Considering the key role of TH-fibers in cooperative synthesis of DA, we assumed the presence of their dopamine regulation. Using double immunolabeling, we showed that D1 and D2 are present in TH-fibers in adult rats, and only D1 in young rats. According to PCR, D1 and D2 are also expressed in neurons of SCN in adult rats and only D1 in young rats. Thus, it was shown for thefirst time that VMAT and D1 are coexpressed in TH-fibers synthesizing L-DOPA in SCN in young and adult rats, and also D2 receptors in adult rats, which suggests vesicular storage and dopamine regulation of L-DOPA secretion, respectively.

МОРФО-ФУНКЦІОНАЛЬНИЙ СТАН ЕПІФІЗА ТА СУПРАХІАЗМАТИЧНОГО ЯДРА ГІПОТАЛАМУСА ЩУРІВ ЗА РІЗНИХ РЕЖИМІВ УВЕДЕННЯ ЕКЗОГЕННОГО МЕЛАТОНІНУ

In modern society increase of digitalization associated with grown exceed level of light at night – a new type of pollution. Presence of light at night inhibited endogenous melatonin synthesis by pineal gland, that influence on circadian system work cycles, so organism oftenbroken regime of wake/sleep, meals, physical activity. Also, a lack of melatonin in some certain time of dayand low melatonin concentration both, were shown take some intervention in diseases development through incorrect regulation of clock-depended genes expression. In connect with this, some latest clinical protocol in therapy or clinical trials of many different pathologies (for example, insomnia, metabolic syndrome, cardiovascular diseases, central nervous and immune system trouble, cancer, viral infection, etc.) include exogenous melatonin usage. As melatonin perform his function via endocrine and paracrine ways in variety types of cell, his application take place in wide range of doses and in different time of day (chronotherapeutic approach). Therefore, important to control state of circadian system central elements – pineal gland (main producer of endogenous melatonin) and suprachiasmatic nucleus (SCN) of hypothalamus (central pacemaker of circadian rhythm) in conditionsof exogenous melatonin treatment. Thus, the main goal of our research were analysis of rats pineal gland and hypothalamic SCN morpho-functional state after different time (morning, evening and continuously with drinking water) melatonin daily administration. Melatonin was administered by gavage for 7 weeks in dose 30 mg/kg 1 h before lights-off (M ZT11, evening), or 1 h after lights-on (M ZT01, morning), or continuously with drinking water during day-night period (MW). After melatonin use only in MW group pineal gland demonstrates changes in morphology (pinealocytes nucleus had mild basophilic color) and morphometric (increased cross-sectional area of the pinealocytes nucleus in compare with control group) analysis data. Besides, some similar changes were observed in SCN: the cross-sectional area of the SCN neurons nucleus grown in case of usage each of regime melatonin administration, while morphology characteristic remains without any alteration. In general, it suggesting about having by melatonin non-inhibiting features in context of circadian system feedback loop and supposing wide potential for melatonin use with absent huge side effect on central elements of above mentioned system.

Seasonal plasticity in GABAA signaling is necessary for restoring phase synchrony in the master circadian clock network.

Annual changes in the environment threaten survival, and numerous biological processes in mammals adjust to this challenge via seasonal encoding by the suprachiasmatic nucleus (SCN). To tune behavior according to day length, SCN neurons display unified rhythms with synchronous phasing when days are short, but will divide into two sub-clusters when days are long. The transition between SCN states is critical for maintaining behavioral responses to seasonal change, but the mechanisms regulating this form of neuroplasticity remain unclear. Here we identify that a switch in chloride transport and GABAA signaling is critical for maintaining state plasticity in the SCN network. Further, we reveal that blocking excitatory GABAA signaling locks the SCN into its long day state. Collectively, these data demonstrate that plasticity in GABAA signaling dictates how clock neurons interact to maintain environmental encoding. Further, this work highlights factors that may influence susceptibility to seasonal disorders in humans.

Role of core circadian clock genes in hormone release and target tissue sensitivity in the reproductive axis

Precise timing in hormone release from the hypothalamus, the pituitary and ovary is critical for fertility. Hormonal release patterns of the reproductive axis are regulated by a feedback loop within the hypothalamic-pituitary-gonadal (HPG) axis. The timing and rhythmicity of hormone release and tissue sensitivity in the HPG axis is regulated by circadian clocks located in the hypothalamus (suprachiasmatic nucleus, kisspeptin and GnRH neurons), the pituitary (gonadotrophs), the ovary (theca and granulosa cells), the testis (Leydig cells), as well as the uterus (endometrium and myometrium). The circadian clocks integrate environmental and physiological signals to produce cell endogenous rhythms generated by a transcriptional-translational feedback loop of transcription factors that are collectively called the “molecular clock”. This review specifically focuses on the contribution of molecular clock transcription factors in regulating hormone release patterns in the reproductive axis, with an emphasis on the female reproductive system. Specifically, we discuss the contributions of circadian rhythms in distinct neuronal populations of the female hypothalamus, the molecular clock in the pituitary and its overall impact on female and male fertility.

Functional Pre- and Postsynaptic Changes between the Retinohypothalamic Tract and Suprachiasmatic Nucleus during Rat Postnatal Development.

The suprachiasmatic nucleus (SCN) is the main brain clock in mammals. The SCN synchronizes to the light-dark cycle through the retinohypothalamic tract (RHT). RHT axons release glutamate to activate AMPA-kainate and N-methyl-D-aspartate (NMDA) postsynaptic receptors in ventral SCN neurons. Stimulation of SCN NMDA receptors is necessary for the activation of the signaling cascades that govern the advances and delays of phase. To our knowledge, no research has been performed to analyze the functional synaptic modifications occurring during postnatal development that prepare the circadian system for a proper synchronization to light at adult ages. Here, we studied the pre- and postsynaptic developmental changes between the unmyelinated RHT-SCN connections. Spontaneous NMDA excitatory postsynaptic currents (EPSCs) were greater in amplitude and frequency at postnatal day 34 (P34) than at P8. Similarly, both quantal EPSCs (miniature NMDA and evoked quantal AMPA-kainate) showed a development-dependent increase at analyzed stages, P3-5, P7-9, and P13-18. Moreover, the electrically evoked NMDA and AMPA-kainate components were augmented with age, although the increment was larger for the latter, and the membrane resting potential was more depolarized at early postnatal ages. Finally, the short-term synaptic plasticity was significantly modified during postnatal development as was the estimated number of quanta released and the initial release probability. All of these synaptic modifications in the unmyelinated RHT-SCN synapses suggest that synchronization to light at adult ages requires developmental changes similar to those that occur in myelinated fast communication systems.

Soriano award lecture: Light, circadian rhythms and sleep: Mechanisms to new therapeutics

Until recently, it seemed inconceivable to most vision researchers and ophthalmologists that there could be an unrecognised class of light sensor (photoreceptor) within the eye. However, by studying how circadian rhythms and sleep are regulated by the dawn/dusk cycle we demonstrated that there exists a “3rd class” of photoreceptor within the eye based upon a small number of photosensitive retinal ganglion cells (pRGCs) that utilise the blue light sensitive photopigment melanopsin (OPN4). The first part of the presentation will consider the discovery and clinical importance of the “third” photoreceptor system of the eye. The second part of the talk will address how these photoreceptors signal light to the molecular clockwork and by what means this pathway is modified by sleep history. There has been remarkable progress in understanding the complex intracellular mechanisms that generate circadian rhythms, the molecular pathways whereby the pRGCs entrain circadian biology and sleep has remained poorly understood. The suprachiasmatic nuclei (SCN) are the site of the primary circadian pacemakers within the mammalian brain. Until recently, the model for entrainment involved a simple linear pathway whereby glutamate release from the pRGCs resulted in Ca2+ influx and raised intracellular cAMP in SCN neurones, which in turn resulted in CREB phosphorylation leading to increased transcription of two key clock genes, Per1 and Per2. This signal then advanced or delayed the molecular clockwork. However, an important feature of entrainment is that circadian responses to light are limited – as typified by jet-lag. Full recovery from jet-lag requires a day for every time-zone crossed. We addressed this issue and have identified and characterized a key role for Salt Inducible Kinase 1 (SIK1) and the CREB-regulated transcription co-activator 1 (CRTC1) in clock re-setting. However, our most recent findings have shown that light entrainment also involves the parallel activation of a Ca2+-ERK1/2-AP-1 signalling pathway. Thus both CRE and AP-1 regulatory elements drive light-induced clock gene expression. In addition, whilst light activation of the Ca2+-ERK1/2-AP-1 signalling pathway increases Per1 and Per2 expression, sleep/wake behaviour alters the effects of light on the clock. Our proposed mechanism suggests that adenosine acts as a signalling molecule that encodes wake duration. Adenosine acts via inhibitory A1 receptors on the SCN to inhibit the Ca2+-ERK1/2-AP-1 signalling pathway, which in turn, reduces the expression of Per1 and Per2. Thus sleep/wake history, encoded by adenosine, reduces the phase shifting effects of light upon the circadian system, altering sleep/wake timing. Finally, the talk will explore how such signalling pathways provide a new target for the regulation of circadian rhythms and the “pharmacological” replacement of light for sleep/wake re-setting in individuals lacking eyes or other individuals with severe circadian rhythm disruption.

Role of Intracellular Na+ in the Regulation of [Ca2+]i in the Rat Suprachiasmatic Nucleus Neurons.

Transmembrane Ca2+ influx is essential to the proper functioning of the central clock in the suprachiasmatic nucleus (SCN). In the rat SCN neurons, the clearance of somatic Ca2+ following depolarization-induced Ca2+ transients involves Ca2+ extrusion via Na+/Ca2+ exchanger (NCX) and mitochondrial Ca2+ buffering. Here we show an important role of intracellular Na+ in the regulation of [Ca2+]i in these neurons. The effect of Na+ loading on [Ca2+]i was determined with the Na+ ionophore monensin and the cardiac glycoside ouabain to block Na+/K+-ATPase (NKA). Ratiometric Na+ and Ca2+ imaging was used to measure the change in [Na+]i and [Ca2+]i, and cell-attached recordings to investigate the effects of monensin and ouabain on spontaneous firing. Our results show that in spite of opposite effects on spontaneous firing and basal [Ca2+], both monensin and ouabain induced Na+ loading, and increased the peak amplitude, slowed the fast decay rate, and enhanced the slow decay phase of 20 mM K+-evoked Ca2+ transients. Furthermore, both ouabain and monensin preferentially enhanced nimodipine-insensitive Ca2+ transients. Together, our results indicate that in the SCN neurons the NKA plays an important role in regulating [Ca2+]i, in particular, associated with nimodipine-insensitive Ca2+ channels.

The Role of Alpha-Tocopherol and Cycloferon in the Regulation of Apoptosis in Neurons of the Hypothalamic Suprachiasmatic Nucleus and Pinealocytes during Stress and Aging

The suprachiasmatic nucleus (SCN) of the hypothalamus and the pineal gland (epiphysis) play an important role not only in the regulation of circadian rhythms but also in the implementation of adaptive responses, including those to various stress factors. Age-related morphofunctional changes in these brain structures, including those associated with increased oxidative stress, exert a significant effect on the organism as a whole. The aim of this work was to explore the dynamics and mechanism of apoptosis in pinealocytes and SCN neurosecretory cells as well as to determine the possibilities of pharmacological correcting this process by an antioxidant alpha-tocopherol and an immunomodulator cycloferon under physiological and immobilization stress conditions in young (2–4-month-old) and aged (30-month-old) Wistar rats. The preparations were administered perorally once a day for 14 days. While the apoptosis level increased with age both in the SCN and the pineal gland, administration of alpha-tocopherol, cycloferon and their combination led to abate this process. It was shown that stress-induced apoptosis in the SCN and the pineal gland proceeds via the p53-dependent pathway, while administration of alpha-tocopherol acetate, cycloferon and their combination decreases the apoptosis level in pinealocytes, suppressing p53 expression both in young and aged animals. In the SCN, no relationship was found between apoptosis and p53 expression levels after administration of the above preparations during stress, suggesting the involvement of different mechanisms.

Disassortative Network Structure Improves the Synchronization between Neurons in the Suprachiasmatic Nucleus.

In mammals, an endogenous clock located in the suprachiasmatic nucleus (SCN) of the brain regulates the circadian rhythms of physiological and behavioral activities. The SCN is composed of about 20,000 neurons that are autonomous oscillators with nonidentical intrinsic periods ranging from 22 h to 28 h. These neurons are coupled through neurotransmitters and synchronized to form a network, which produces a robust circadian rhythm of a uniform period. The neurons, which are the nodes in the network, are known to be heterogeneous in their characteristics, which is reflected in different phenotypes and different functionality. This heterogeneous nature of the nodes of the network leads to the question as to whether the structure of the SCN network is assortative or disassortative. Thus far, the disassortativity of the SCN network has not been assessed and neither have its effects on the collective behaviors of the SCN neurons. In the present study, we build a directed SCN network composed of hundreds of neurons for a single slice using the method of transfer entropy, based on the experimental data. Then, we measured the synchronization degree as well as the disassortativity coefficient of the network structure (calculated by either the out-degrees or the in-degrees of the nodes) and found that the network of the SCN is a disassortative network. Furthermore, a positive relationship is observed between the synchronization degree and disassortativity of the network, which is confirmed by simulations of our modeling. Our finding suggests that the disassortativity of the network structure plays a role in the synchronization between SCN neurons; that is, the synchronization degree increases with the increase of the disassortativity, which implies that a more heterogeneous coupling in the network of the SCN is important for proper function of the SCN.

Spatiotemporal expression pattern of PERIOD 1 and PERIOD 2 in the mouse SCN is dependent on VIP receptor 2 signaling.

Neurons of the hypothalamic suprachiasmatic nucleus (SCN) express clock genes, which regulate their own transcription and generate daily output signals driving circadian rhythmic behavior and physiology. The neuropeptide VIP and its specific receptor, the VPAC2 receptor, are important for synchronization of clock neurons. In the present study, we characterized PER1 and PER2 expressing neurons in wild-type and VPAC2-deficient mice. We found evidence for distinct spatiotemporal circadian oscillation in the expression of the PER genes in two separate clusters of SCN neurons. In wild-type mice corresponding to the SCN shell and ventral core, high expression of PER was found at lights-off most likely representing an evening clock (E-clock). In another smaller cluster of neurons located in the central core of the SCN, PER expression peaks in antiphase at lights-on and could represent a morning clock (M-clock). BMAL1 immunoreactivity was found to be expressed in antiphase to PER in M and E neurons, respectively. PER was found in 98% of neurons expressing vasopressin (AVP) and in 92% of VIP neurons. The chemotype of M neurons was not identified. M but not E cells were responsive to long but not short photoperiods. The expression of the VPAC2 receptor was found in both M and E cells, and VPAC2-deficient mice displayed markedly blunted PER expression in both cell clusters of the SCN. Conclusion: These observations support the existence of M and E clocks involved in circadian and seasonal adaptation, which seem dependent on intact VIP/VPAC2 signaling in the SCN.

Diurnal properties of voltage-gated Ca2+ currents in suprachiasmatic nucleus and roles in action potential firing.

Circadian oscillations in spontaneous action potential firing in the suprachiasmatic nucleus (SCN) translate time-of-day throughout the mammalian brain. The ion channels that regulate the circadian pattern of SCN firing have not been comprehensively identified. Ca2+ channels regulate action potential activity across many types of excitable cells, and the activity of L-, N-, P/Q- and R-type channels are required for normal daytime firing frequency in SCN neurons and circuit rhythms. Only the L-type Ca2+ current exhibits a day versus night difference in current magnitude, providing insight into the mechanism that produces rhythmic action potential firing in SCN. The mammalian circadian clock encodes time via rhythmic action potential activity in the suprachiasmatic nucleus (SCN) of the hypothalamus, which governs daily rhythms in physiology and behaviour. SCN neurons exhibit 24h oscillations in spontaneous firing, with higher firing during day compared to night. Several ionic currents have been identified that regulate SCN firing, including voltage-gated Ca2+ currents, but the circadian regulation of distinct voltage-gated Ca2+ channel (VGCC) components has not been comprehensively addressed. In this study, whole-cell L- (nimodipine-sensitive), N- and P/Q- (ω-agatoxin IVA, ω-conotoxin GVIA, ω-conotoxin MVIIC-sensitive), R- (Ni2+ -sensitive) and T-type (TTA-P2-sensitive) currents were recorded from day and night SCN slices. Using standard voltage protocols, Ni2+ -sensitive currents comprised the largest proportion of total VGCC current, followed by nimodipine-, ω-agatoxin IVA-, ω-conotoxin GVIA- and TTA-P2-sensitive currents. Only the nimodipine-sensitive current exhibited a diurnal difference in magnitude, with daytime current larger than night. No diurnal variation was observed for the other Ca2+ current subtypes. The difference in nimodipine-sensitive current was due to larger peak current activated during the day, not differences in inactivation, and was eliminated by Bay K8644. Blocking L-type channels decreased firing selectively during the day, consistent with higher current magnitudes, and reduced SCN circuit rhythmicity recorded by multi-electrode arrays. Yet blocking N-, P/Q- and R-type channels also decreased daytime firing, with little effect at night, and decreased circuit rhythmicity. These data identify a unique diurnal regulation of L-type current among the major VGCC subtypes in SCN neurons, but also reveal that diurnal modulation is not required for time-of-day-specific effects on firing and circuit rhythmicity.

GABA in the suprachiasmatic nucleus refines circadian output rhythms in mice

In mammals, the circadian rhythms are regulated by the central clock located in the hypothalamic suprachiasmatic nucleus (SCN), which is composed of heterogeneous neurons with various neurotransmitters. Among them an inhibitory neurotransmitter, γ-Amino-Butyric-Acid (GABA), is expressed in almost all SCN neurons, however, its role in the circadian physiology is still unclear. Here, we show that the SCN of fetal mice lacking vesicular GABA transporter (VGAT−/−) or GABA synthesizing enzyme, glutamate decarboxylase (GAD65−/−/67−/−), shows burst firings associated with large Ca2+ spikes throughout 24 hours, which spread over the entire SCN slice in synchrony. By contrast, circadian PER2 rhythms in VGAT−/− and GAD65−/−/67−/− SCN remain intact. SCN-specific VGAT deletion in adult mice dampens circadian behavior rhythm. These findings indicate that GABA in the fetal SCN is necessary for refinement of the circadian firing rhythm and, possibly, for stabilizing the output signals, but not for circadian integration of multiple cellular oscillations.

Connexin30 and Connexin43 show a time-of-day dependent expression in the mouse suprachiasmatic nucleus and modulate rhythmic locomotor activity in the context of chronodisruption

BackgroundThe astroglial connexins Cx30 and Cx43 contribute to many important CNS functions including cognitive behaviour, motoric capacity and regulation of the sleep-wake cycle. The sleep wake cycle, is controlled by the circadian system. The central circadian rhythm generator resides in the suprachiasmatic nucleus (SCN). SCN neurons are tightly coupled in order to generate a coherent circadian rhythm. The SCN receives excitatory glutamatergic input from the retina which mediates entrainment of the circadian system to the environmental light-dark cycle. Connexins play an important role in electric coupling of SCN neurons and astrocytic-neuronal signalling that regulates rhythmic SCN neuronal activity. However, little is known about the regulation of Cx30 and Cx43 expression in the SCN, and the role of these connexins in light entrainment of the circadian system and in circadian rhythm generation.MethodsWe analysed time-of-day dependent as well as circadian expression of Cx30 and Cx43 mRNA and protein in the mouse SCN by means of qPCR and immunohistochemistry. Moreover, we analysed rhythmic spontaneous locomotor activity in mice with a targeted deletion of Cx30 and astrocyte specific deletion of Cx43 (DKO) in different light regimes by means of on-cage infrared detectors.ResultsFluctuation of Cx30 protein expression is strongly dependent on the light-dark cycle whereas fluctuation of Cx43 protein expression persisted in constant darkness. DKO mice entrained to the light-dark cycle. However, re-entrainment after a phase delay was slightly impaired in DKO mice. Surprisingly, DKO mice were more resilient to chronodisruption.ConclusionCircadian fluctuation of Cx30 and Cx43 protein expression in the SCN is differently regulated. Cx30 and astroglial Cx43 play a role in rhythm stability and re-entrainment under challenging conditions.

Musashi-2 and related stem cell proteins in the mouse suprachiasmatic nucleus and their potential role in circadian rhythms

BackgroundThe suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains the master circadian clock of the body and an unusually large number of cells expressing stem cell-related proteins. These seemingly undifferentiated cells may serve in entrainment of the SCN circadian clock to light cycles or allow it to undergo neural plasticity important for modifying its rhythmic output signals. These cells may also proliferate and differentiate into neurons or glia in response to episodic stimuli or developmental events requiring alterations in the SCN’s control of physiology and behavior. ProblemTo characterize expression of stem cell related proteins in the SCN and the effects of stem-like cells on circadian rhythms. MethodsExplant cultures of mouse SCN were maintained in medium designed to promote survival and growth of stem cells but not neuronal cells. Several stem cell marker proteins including SRY-box containing gene 2 (SOX2), nestin, vimentin, octamer-binding protein 4 (OCT4), and Musashi RNA-binding protein 2 (MSI2) were identified by immunocytochemistry in histological sections from adult mouse SCN and in cultures of microdissected SCN. A bioinformatics analysis located potential SCN targets of MSI2 and related RNA-binding proteins. ResultsCells expressing stem cell markers proliferated in culture. Immunostained brain sections and bioinformatics supported the view that MSI2 regulates immature properties of SCN neurons, potentially providing flexibility in SCN neural circuits. Explant cultures had ongoing mitotic activity, indicated by proliferating-cell nuclear antigen, and extensive cell loss shown by propidium iodide staining. Cells positive for vasoactive intestinal polypeptide (VIP) that are highly enriched in the SCN were diminished in explant cultures. Despite neuronal cell loss, tissue remained viable for over 7 weeks in culture, as shown by bioluminescence imaging of explants prepared from SCN of Per1::luc transgenic mice. The circadian rhythm in SCN gene expression persisted in brain slice cultures in stem cell medium. Prominent, widespread expression of RNA-binding protein MSI2 supported the importance of posttranscriptional regulation in SCN functions and provided further evidence of stem-like cells. ConclusionThe results show that the SCN retains properties of immature neurons and these properties persist in culture conditions suitable for stem cells, where the SCN stem-like cells also proliferate. These properties may allow adaptive circadian rhythm adjustments. Further exploration should examine stem-like cells of the SCN in vivo, how they may affect circadian rhythms, and whether MSI2 serves as a master regulator of SCN stem-like properties.

From clock to functional pacemaker.

In mammals, the central pacemaker that coordinates 24‐hr rhythms is located in the suprachiasmatic nucleus (SCN). Individual neurons of the SCN have a molecular basis for rhythm generation and hence, they function as cell autonomous oscillators. Communication and synchronization among these neurons are crucial for obtaining a coherent rhythm at the population level, that can serve as a pace making signal for brain and body. Hence, the ability of single SCN neurons to produce circadian rhythms is equally important as the ability of these neurons to synchronize one another, to obtain a bona fide pacemaker at the SCN tissue level. In this chapter we will discuss the mechanisms underlying synchronization, and plasticity herein, which allows adaptation to changes in day length. Furthermore, we will discuss deterioration in synchronization among SCN neurons in aging, and gain in synchronization by voluntary physical activity or exercise.

The Mammalian Circadian Timing System and the Suprachiasmatic Nucleus as Its Pacemaker.

The past twenty years have witnessed the most remarkable breakthroughs in our understanding of the molecular and cellular mechanisms that underpin circadian (approximately one day) time-keeping. Across model organisms in diverse taxa: cyanobacteria (Synechococcus), fungi (Neurospora), higher plants (Arabidopsis), insects (Drosophila) and mammals (mouse and humans), a common mechanistic motif of delayed negative feedback has emerged as the Deus ex machina for the cellular definition of ca. 24 h cycles. This review will consider, briefly, comparative circadian clock biology and will then focus on the mammalian circadian system, considering its molecular genetic basis, the properties of the suprachiasmatic nucleus (SCN) as the principal circadian clock in mammals and its role in synchronising a distributed peripheral circadian clock network. Finally, it will consider new directions in analysing the cell-autonomous and circuit-level SCN clockwork and will highlight the surprising discovery of a central role for SCN astrocytes as well as SCN neurons in controlling circadian behaviour.

SOX2-Dependent Transcription in Clock Neurons Promotes the Robustness of the Central Circadian Pacemaker

Clock neurons within the mammalian suprachiasmatic nuclei (SCN) encode circadian time using interlocked transcription-translation feedback loops (TTFLs) that drive rhythmic gene expression. However, the contributions of other transcription factors outside of the circadian TTFLs to the functionality of the SCN remain obscure. Here, we report that the stem and progenitor cell transcription factor, sex-determining region Y-box 2 (SOX2), is expressed inadult SCN neurons and positively regulates transcription of the core clock gene, Period2. Mice lacking SOX2 selectively in SCN neurons display imprecise, poorly consolidated behavioral rhythms that do not entrain efficiently to environmental light cycles and that are highly susceptible to constant light-induced arrhythmicity. RNA sequencing revealed that Sox2 deficiency alters the SCN transcriptome, reducing the expression of core clock genes and neuropeptide-receptor systems. By defining the transcriptional landscape within SCN neurons, SOX2 enables the generation of robust, entrainable circadian rhythms that accurately reflect environmental time.

The Network Mechanism of the Central Circadian Pacemaker of the SCN: Do AVP Neurons Play a More Critical Role Than Expected?

The suprachiasmatic nucleus (SCN) functions as the central circadian pacemaker in mammals and entrains to the environmental light/dark cycle. It is composed of multiple types of GABAergic neurons, and interneuronal communications among these neurons are essential for the circadian pacemaking of the SCN. However, the mechanisms underlying the SCN neuronal network remain unknown. This review will provide a brief overview of the current knowledge concerning the differential roles of multiple neuropeptides and neuropeptide-expressing neurons in the SCN, especially focusing on the emerging roles of arginine vasopressin-producing neurons uncovered by recent studies utilizing neuron type-specific genetic manipulations in mice.

Calcium Channels Contributing to Action Potential Firing and Rhythms in the Circadian Clock

Mammalian circadian rhythms are timed by suprachiasmatic nucleus (SCN) neurons, which generate a ∼24-hour time signal by firing spontaneous action potentials (APs) at a higher frequency during the day versus the night. The roles of Ca2+ channels in regulating AP firing in the SCN have not been extensively studied. To identify which Ca2+ channels contribute to the circadian regulation of AP frequency, we tested the effects of inhibitors of VGCCs (Cd2+), L-(nimodipine, Nim), N-(ω-conotoxinGVIA, GVIA), P/Q-(ω-agatoxinIVA, Aga), R-(Ni2+), and T-(TTAP2) type Ca2+ channels, ryanodine receptors (dantrolene, Dan) and SERCA (cyclopiazonic acid, CPA) on extracellular day and night AP frequencies from acute SCN slices. Compared to control, daytime AP frequency decreased in Cd2+ (-85%), Nim (−28%), GVIA (−65%), Aga (−69%), Ni2+ (-46%), Dan (−31%), and CPA (−27%). Nighttime AP frequency also decreased in Cd2+ (-63%) but increased in Dan (+113%) and CPA (+125%). The chronic effect of these inhibitors on AP rhythmicity was determined using 3 days of baseline firing and 3 days post-drug application from organotypic SCNs on multi-electrode arrays. Compared to baseline, the percentage of rhythmic recordings decreased in Nim (−16±6%), GVIA (−16±6%), Aga (−29±5%) and Ni2+ (−32±10%). Of the rhythmic recordings remaining, the χ2 amplitude decreased with Nim (−55%) and Aga (−48%) and relative power (FFT) decreased with Nim (−57%). Period increased in Dan (+5%), although Dan and CPA did not affect other rhythmic parameters. Our data suggests L-, N-, P/Q- and R-TCCs all contribute to daytime AP frequency and circadian AP rhythms. The strongest influence is contributed by LTCCs, consistent with evidence that LTCC currents are diurnally modulated. In contrast, Ca2+i stores contribute to daytime and nighttime AP frequency but only a limited influence on AP rhythmicity is revealed in this study.