ABSTRACT
We have previously developed a 3D video tracking system which enables us to analyze long-term quantitative analysis of gene expression in freely moving mice. In the present study, we improved 3D video tracking and developed a system that analyzes more detailed behavioral data. We succeeded in simultaneously analyzing sleep-wake, feeding, and drinking behavior rhythms in the same individual using our tracking system. This system will make it possible to measure gene expression in each tissue in vivo in real time in relation to the various behavioral rhythms mentioned above.
ABSTRACT
Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) is widely expressed in the brain and is involved in various functions, including memory formation, mood and sleep. We previously reported that CaMKIIα is involved in the circadian molecular clock. Mice lacking functional CaMKIIα (K42R mice) exhibited a gradual increase in activity time (α decompression) of running-wheel (RW) activity due to a lengthened circadian period (τ) of activity offset under constant darkness (DD). In the present study, to investigate the functional roles of CaMKIIα in behavioural rhythms, we measured RW and general movements simultaneously under prolonged DD. Tau became longer as the relative intensity of behaviour activity within an activity time shifted from activity onset towards activity offset. In some K42R mice, α was gradually expanded with a marked reduction of RW activity, while general movements persisted without noticeable decline, which was followed by an abrupt shortening of α (α compression) with differential phase shifts of the activity onset and offset and recovery of RW activity. These results suggest that an internal coupling between the oscillators controlling activity onset and offset is bidirectional but with different strengths. The α compression occurred recurrently in 38% of K42R mice examined with an average interval of 37 days in association with attenuation of RW activity but never in the wild-type (WT) mice. Consistent with behavioural rhythms, the circadian period of the PER2::LUC rhythm in the cultured suprachiasmatic nucleus (SCN) slice was significantly longer in K42R than in WT. These findings are best interpreted by assuming that a loss of functional CaMKIIα attenuates the coupling between the onset and offset oscillators.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Circadian Rhythm , Suprachiasmatic Nucleus , Animals , Male , Mice , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Circadian Clocks/physiology , Circadian Rhythm/physiology , Mice, Inbred C57BL , Motor Activity/physiology , Period Circadian Proteins/metabolism , Period Circadian Proteins/genetics , Suprachiasmatic Nucleus/physiology , Suprachiasmatic Nucleus/metabolismABSTRACT
It has been 50 years since the suprachiasmatic nucleus (SCN) was first identified as the central circadian clock and 25 years since the last overview of developments in the field was published in the Journal of Biological Rhythms. Here, we explore new mechanisms and concepts that have emerged in the subsequent 25 years. Since 1997, methodological developments, such as luminescent and fluorescent reporter techniques, have revealed intricate relationships between cellular and network-level mechanisms. In particular, specific neuropeptides such as arginine vasopressin, vasoactive intestinal peptide, and gastrin-releasing peptide have been identified as key players in the synchronization of cellular circadian rhythms within the SCN. The discovery of multiple oscillators governing behavioral and physiological rhythms has significantly advanced our understanding of the circadian clock. The interaction between neurons and glial cells has been found to play a crucial role in regulating these circadian rhythms within the SCN. Furthermore, the properties of the SCN network vary across ontogenetic stages. The application of cell type-specific genetic manipulations has revealed components of the functional input-output system of the SCN and their correlation with physiological functions. This review concludes with the high-risk effort of identifying open questions and challenges that lie ahead.
Subject(s)
Circadian Rhythm , Neuropeptides , Circadian Rhythm/physiology , Neuropeptides/metabolism , Suprachiasmatic Nucleus/physiology , Vasoactive Intestinal Peptide/metabolism , Gastrin-Releasing Peptide/metabolismABSTRACT
The sleep-wake cycle of human subjects was artificially split into two episodes by imposing an 8-h light and 4-h dark cycle (LD 8:4) twice a day for 7 days, which was followed by a 3-day free-running session. Sleep was permitted only in the dark period. The subjects in the ordinary group were exposed to ordinary light (ca. 500 lx) in the 8-h light period, and those in the bright light group to bright (ca. 5,000 lx) and ordinary light alternatively with bright light after the first dark period (2400-400). Split sleeps persisted in the free-running session with the major episode around the first dark period and the minor episode around the second dark period. By contrast, circadian melatonin rhythm in the free-running session significantly phase delayed in the ordinary light group, but phase advanced in the bright light group, keeping the melatonin rhythm unsplit. The length of nocturnal melatonin secretion (NMS) was significantly shortened in the bright light group. Interestingly, the falling phase of NMS advanced significantly further than the rising phase. Such a difference was not detected in the ordinary light group. Similar differences were observed in the body temperature rhythm. These findings indicated oscillatory mechanisms underlying split sleeps distinct from the circadian pacemaker and suggested an involvement of different circadian oscillators in the rising and falling phases of NMS, which is consistent with the dual oscillator model proposed for the circadian system of nocturnal rodents.NEW & NOTEWORTHY The present study demonstrated that human sleep was separated into two essentially identical components, which persisted under constant conditions, suggesting circadian oscillator underlying split-sleep episodes. The study also indicated differential light sensitivities in the rising and falling phases of circadian melatonin rhythm, indicating the involvement of two different oscillators. These results consisted of the evening and morning dual-oscillator hypothesis for the circadian pacemaker and the hierarchical model for the pacemaker and sleep-wake cycle.
Subject(s)
Melatonin , Humans , Circadian Rhythm/physiology , Sleep/physiology , Body Temperature/physiology , LightABSTRACT
Circadian rhythms and sleep-wake cycles were measured in volunteers staying singly in temporal isolation unit where they were exposed to artificial short and long light-dark (LD) cycles for 7 days. The long day consisted of 16-h light and 8-h dark (LD 16:8) and the short day consisted of 8-h light and 16-h dark (LD 8:16). During the light period, bright light of approximately 5,000 lux was given from the ceiling and during the dark period there was no illumination. Sleep was monitored by bed sensors, wrist actiwatch, and polysomnography (PSG) on the first and last nights of the schedule. Sleep length was significantly longer under LD 8:16 than under LD 16:8 and the sleep quality estimated by PSG was worse under LD 8:16 than under LD 16:8, which were comparable to natural seasonality in sleep. The circadian rhythm in plasma melatonin was measured in dim light (10 lux) before and after the LD exposures. The nocturnal melatonin secretion (NMS) was significantly longer after LD 8:16 than after LD 16:8 due to differential phase shifts of the rising and falling phases of NMS. After LD 8:16, the falling phase was much advanced than the rising phase, whereas after LD 16:8 the rising phase was much delayed than the falling phase, resulting in the NMS compression. These results indicate that the light sensitivity in terms of phase shifting is different in the two circadian phases, supporting a dual oscillator hypothesis with different phase-response curves for light in the human circadian system.NEW & NOTEWORTHY The present study demonstrated differential light responsiveness of the rising and falling phases of nocturnal melatonin secretion in human subjects exposed to artificial long (LD 16:8) and short days (LD 8:16) and suggested the involvement of different oscillators under these phases. The findings well mimicked the seasonality of the circadian rhythms in nature and consisted with the evening/morning dual oscillator hypothesis proposed originally for nocturnal rodents, providing a new concept for the human circadian system.
ABSTRACT
Effects of a fixed single meal per day were examined on the circadian pacemaker and sleep-wake cycle in subjects under temporal isolation. When the time of single meal was allowed to take at any time of day (ad-lib meal), the sleep-wake cycle as well as the circadian rhythms in plasma melatonin, cortisol, and core body temperature were significantly phase-delayed in 8 days. On the other hand, when the time of meal was fixed at 1800 h in local time (RF meal), the phase-shift of sleep-wake cycle was not significant while those of the circadian rhythms were significant. The differential effects of a fixed single meal schedule were confirmed in most individual subjects. There was no evidence for the prefeeding increase in plasma cortisol and leptin levels under the fixed single meal schedule. The plasma ghrelin level was apparently high before meal in both ad-lib and RF meal groups, which was, however, likely sculptured by a nonspecific prandial drop and gradual increase after meal intake. Single meal augmented the prandial increase of plasma insulin levels by four to five times. These findings indicate that a single meal at a fixed time of the day during the subjective day failed to prevent the human circadian pacemaker but prevented the sleep-wake cycle from free running for at least 8 days under temporal isolation, suggesting that mealtime was a potent nonphotic time cue for the human sleep-wake cycle.
Subject(s)
Melatonin , Wakefulness , Body Temperature/physiology , Circadian Rhythm/physiology , Humans , Hydrocortisone , Meals , Sleep/physiology , Wakefulness/physiologyABSTRACT
We report two cases who had been diagnosed with major depression, but found to have periodic limb movement disorder (PLMD) or restless legs syndrome (RLS) as major disorder. Both patients had difficulties in occupational and/or daily lives. In neither case, antidepressants were effective in symptom remission. In contrast, rotigotine transdermal patch was effective not only for core symptoms of PLMD or RLS but also for accompanying depressive symptoms. Since PLMD and RLS are associated with dopaminergic dysfunction etiologically, a dopamine receptor agonist rotigotine might be a good choice for patients with PLMD or RLS accompanying depression.
ABSTRACT
[This corrects the article DOI: 10.3389/fnins.2021.650154.].
ABSTRACT
The food-entrainable oscillator, which underlies the prefeeding activity peak developed by restricted daily feeding (RF) in rodents, does not depend on the circadian pacemaker in the suprachiasmatic nucleus (SCN) or on the known clock genes. In the present study, to clarify the roles of SCN circadian pacemaker and nutrient conditions on the development of prefeeding activity peak, RF of 3-h daily feeding was imposed on four groups of adult male mice for 10 cycles at different circadian times, zeitgeber time (ZT)2, ZT8, ZT14, and ZT20, where ZT0 is the time of lights-on in LD12:12. Seven days after the termination of RF session with ad libitum feeding in between, total food deprivation (FD) for 72 h was imposed. Wheel-running activity and core body temperature were measured throughout the experiment. Immediately after the RF or FD session, the PER2::LUC rhythms were measured in the cultured SCN slices and peripheral tissues. Not only the buildup process and magnitude of the prefeeding activity peak, but also the percentages of nocturnal activity and hypothermia developed under RF were significantly different among the four groups, indicating the involvement of light entrained circadian pacemaker. The buildup of prefeeding activity peak was accomplished by either phase-advance or phase-delay shifts (or both) of activity bouts comprising a nocturnal band. Hypothermia under FD was less prominent in RF-exposed mice than in naïve counterparts, indicating that restricted feeding increases tolerance to caloric restriction as well as to the heat loss mechanism. RF phase-shifted the peripheral clocks but FD did not affect the clocks in any tissue examined. These findings are better understood by assuming multiple bout oscillators, which are located outside the SCN and directly drive activity bouts uncoupled from the circadian pacemaker by RF or hypothermia.
Subject(s)
Circadian Rhythm , Animals , Feeding Behavior , Food , Male , Mice , Suprachiasmatic NucleusABSTRACT
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.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Circadian Rhythm/physiology , Homeodomain Proteins/metabolism , Repressor Proteins/metabolism , Suprachiasmatic Nucleus/metabolism , Transcription Factors/metabolism , Animals , Animals, Newborn , Basic Helix-Loop-Helix Transcription Factors/genetics , Cryptochromes/genetics , Female , Homeodomain Proteins/genetics , Male , Mice , Mice, Knockout , Repressor Proteins/genetics , Transcription Factors/geneticsABSTRACT
Biological neural networks operate at several levels of granularity, from the individual neuron to local neural circuits to networks of thousands of cells. The daily oscillation of the brain's master clock in the suprachiasmatic nucleus (SCN) rests on a yet to be identified network of connectivity among its â¼20,000 neurons. The SCN provides an accessible model to explore neural organization at several levels of organization. To relate cellular to local and global network behaviors, we explore network topology by examining SCN slices in three orientations using immunochemistry, light and confocal microscopy, real-time imaging, and mathematical modeling. Importantly, the results reveal small local groupings of neurons that form intermediate structures, here termed "phaseoids," which can be identified through stable local phase differences of varying magnitude among neighboring cells. These local differences in phase are distinct from the global phase relationship, namely that between individual cells and the mean oscillation of the overall SCN. The magnitude of the phaseoids' local phase differences is associated with a global phase gradient observed in the SCN's rostral-caudal extent. Modeling results show that a gradient in connectivity strength can explain the observed gradient of phaseoid strength, an extremely parsimonious explanation for the heterogeneous oscillatory structure of the SCN.
Subject(s)
Neurons , Suprachiasmatic Nucleus , Anisotropy , Circadian RhythmABSTRACT
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.
ABSTRACT
The mammalian central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN contains multiple circadian oscillators which synchronize with each other via several neurotransmitters. Importantly, an inhibitory neurotransmitter, γ-amino butyric acid (GABA), is expressed in almost all SCN neurons. In this review, we discuss how GABA influences circadian rhythms in the SCN. Excitatory and inhibitory effects of GABA may depend on intracellular Cl- concentration, in which several factors such as day-length, time of day, development, and region in the SCN may be involved. GABA also mediates oscillatory coupling of the circadian rhythms in the SCN. Recent genetic approaches reveal that GABA refines circadian output rhythms, but not circadian oscillations in the SCN. Since several efferent projections of the SCN have been suggested, GABA might work downstream of neuronal pathways from the SCN which regulate the temporal order of physiology and behavior.
Subject(s)
Circadian Rhythm/physiology , Neurons/metabolism , Period Circadian Proteins/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Humans , Hypothalamus/metabolism , Suprachiasmatic Nucleus/metabolismABSTRACT
The mammalian circadian system is composed of a central clock situated in the hypothalamic suprachiasmatic nucleus (SCN) and peripheral clocks of each tissue and organ in the body. While much has been learned about the pre- and postnatal development of the circadian system, there are still many unanswered questions about how and when cellular clocks start to tick and form the circadian system. Most SCN neurons contain a cell-autonomous circadian clock with individual specific periodicity. Therefore, the network of cellular oscillators is critical for the coherent rhythm expression and orchestration of the peripheral clocks by the SCN. The SCN is the only circadian clock entrained by an environmental light-dark cycle. Photic entrainment starts postnatally, and the SCN starts to function gradually as a central clock that controls physiological and behavioral rhythms during postnatal development. The SCN exhibits circadian rhythms in clock gene expression from the embryonic stage throughout postnatal life and the rhythm phenotypes remain basically unchanged. However, the disappearance of coherent circadian rhythms in cryptochrome-deficient SCN revealed changes in the SCN networks that occur in postnatal weeks 2-3. The SCN network consists of multiple clusters of cellular circadian rhythms that are differentially integrated by the vasoactive intestinal polypeptide and arginine vasopressin signaling depending on the period of postnatal development.
Subject(s)
Circadian Clocks , Animals , Circadian Rhythm , Cryptochromes , Photoperiod , Suprachiasmatic NucleusABSTRACT
Circadian rhythms in Per1, PER2 expression and intracellular Ca2+ were measured from a solitary SCN neuron or glial cell which was physically isolated from other cells. Dispersed cells were cultured on a platform of microisland (100-200 µm in diameter) in a culture dish. Significant circadian rhythms were detected in 57.1% for Per1 and 70.0% for PER2 expression. When two neurons were located on the same island, the circadian rhythms showed desynchronization, indicating a lack of oscillatory coupling. Circadian rhythms were also detected in intracellular Ca2+ of solitary SCN neurons. The ratio of circadian positive neurons was significantly larger without co-habitant of glial cells (84.4%) than with it (25.0%). A relatively large fraction of SCN neurons generates the intrinsic circadian oscillation without neural or humoral networks. In addition, glial cells seem to interrupt the expression of the circadian rhythmicity of intracellular Ca2+ under these conditions.
Subject(s)
Calcium/metabolism , Circadian Rhythm , Period Circadian Proteins/metabolism , Suprachiasmatic Nucleus Neurons/metabolism , Animals , Cells, Cultured , Mice , Mice, Inbred C57BL , Single-Cell Analysis , Suprachiasmatic Nucleus Neurons/cytologyABSTRACT
Circadian rhythms are generated by interlocked transcriptional-translational negative feedback loops (TTFLs), the molecular process implemented within a cell. The contributions, weighting and balancing between the multiple feedback loops remain debated. Dissociated, free-running dynamics in the expression of distinct clock genes has been described in recent experimental studies that applied various perturbations such as slice preparations, light pulses, jet-lag, and culture medium exchange. In this paper, we provide evidence that this "presumably transient" dissociation of circadian gene expression oscillations may occur at the single-cell level. Conceptual and detailed mechanistic mathematical modeling suggests that such dissociation is due to a weak interaction between multiple feedback loops present within a single cell. The dissociable loops provide insights into underlying mechanisms and general design principles of the molecular circadian clock.
Subject(s)
Circadian Clocks/genetics , Circadian Rhythm/genetics , Animals , Computational Biology , Feedback , Gene Expression Regulation/genetics , Humans , Mice , Models, Genetic , Single-Cell Analysis , Suprachiasmatic Nucleus Neurons/cytologyABSTRACT
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.
Subject(s)
Circadian Rhythm/physiology , Suprachiasmatic Nucleus/physiology , gamma-Aminobutyric Acid/physiology , Animals , Calcium/metabolism , Glutamate Decarboxylase/physiology , Mice , Period Circadian Proteins/physiology , Signal Transduction/physiology , Vesicular Inhibitory Amino Acid Transport Proteins/physiologyABSTRACT
Circadian rhythms are controlled by transcriptional feedback loops of clock genes and proteins. The stability of clock proteins is regulated by post-translational modification, such as phosphorylation by kinases. In particular, casein kinase I (CKI) phosphorylates the PER protein to regulate proteasomal degradation and nuclear localization. Therefore, CKI inhibition can modulate mammalian circadian rhythms. In the present study, we have developed novel CKIα and CKIδ dual inhibitors by extensive structural modification of N9 and C2 position of longdaysin. We identified NCC007 that showed stronger period effects (0.32 µM for 5 h period lengthening) in a cell-based circadian assay. The following in vitro kinase assay showed that NCC007 inhibited CKIα and CKIδ with an IC50 of 1.8 and 3.6 µM. We further demonstrated that NCC007 lengthened the period of mouse behavioral rhythms in vivo. Thus, NCC007 is a valuable tool compound to control circadian rhythms through CKI inhibition.
Subject(s)
Adenine/analogs & derivatives , Casein Kinase Ialpha/antagonists & inhibitors , Circadian Rhythm/drug effects , Enzyme Inhibitors/pharmacology , Adenine/chemical synthesis , Adenine/metabolism , Adenine/pharmacology , Animals , Binding Sites , Casein Kinase Ialpha/chemistry , Casein Kinase Ialpha/metabolism , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Humans , Male , Mice, Inbred C57BL , Molecular Docking Simulation , Protein BindingABSTRACT
Circadian clocks are autonomous oscillators driving daily rhythms in physiology and behavior. In mammals, a network of coupled neurons in the suprachiasmatic nucleus (SCN) is entrained to environmental light-dark cycles and orchestrates the timing of peripheral organs. In each neuron, transcriptional feedbacks generate noisy oscillations. Coupling mediated by neuropeptides such as VIP and AVP lends precision and robustness to circadian rhythms. The detailed coupling mechanisms between SCN neurons are debated. We analyze organotypic SCN slices from neonatal and adult mice in wild-type and multiple knockout conditions. Different degrees of rhythmicity are quantified by pixel-level analysis of bioluminescence data. We use empirical orthogonal functions (EOFs) to characterize spatio-temporal patterns. Simulations of coupled stochastic single cell oscillators can reproduce the diversity of observed patterns. Our combination of data analysis and modeling provides deeper insight into the enormous complexity of the data: (1) Neonatal slices are typically stronger oscillators than adult slices pointing to developmental changes of coupling. (2) Wild-type slices are completely synchronized and exhibit specific spatio-temporal patterns of phases. (3) Some slices of Cry double knockouts obey impaired synchrony that can lead to co-existing rhythms ("splitting"). (4) The loss of VIP-coupling leads to desynchronized rhythms with few residual local clusters. Additional information was extracted from co-culturing slices with rhythmic neonatal wild-type SCNs. These co-culturing experiments were simulated using external forcing terms representing VIP and AVP signaling. The rescue of rhythmicity via co-culturing lead to surprising results, since a cocktail of AVP-antagonists improved synchrony. Our modeling suggests that these counter-intuitive observations are pointing to an antagonistic action of VIP and AVP coupling. Our systematic theoretical and experimental study shows that dual coupling mechanisms can explain the astonishing complexity of spatio-temporal patterns in SCN slices.
Subject(s)
Arginine Vasopressin/metabolism , Circadian Rhythm/physiology , Vasoactive Intestinal Peptide/metabolism , Animals , Arginine Vasopressin/physiology , Circadian Clocks/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/physiology , Neuropeptides/metabolism , Period Circadian Proteins/metabolism , Signal Transduction , Suprachiasmatic Nucleus/physiology , Vasoactive Intestinal Peptide/physiologyABSTRACT
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.