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1.
iScience ; 24(10): 103142, 2021 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-34632336

RESUMO

Circadian rhythms persist in almost all organisms and are crucial for maintaining appropriate timing in physiology and behaviour. Here, we describe a mouse mutant where the central mammalian pacemaker, the suprachiasmatic nucleus (SCN), has been genetically ablated by conditional deletion of the transcription factor Zfhx3 in the developing hypothalamus. Mutants were arrhythmic over the light-dark cycle and in constant darkness. Moreover, rhythms of metabolic parameters were ablated in vivo although molecular oscillations in the liver maintained some rhythmicity. Despite disruptions to SCN cell identity and circuitry, mutants could still anticipate food availability, yet other zeitgebers - including social cues from cage-mates - were ineffective in restoring rhythmicity although activity levels in mutants were altered. This work highlights a critical role for Zfhx3 in the development of a functional SCN, while its genetic ablation further defines the contribution of SCN circuitry in orchestrating physiological and behavioral responses to environmental signals.

2.
J Neurosci ; 41(41): 8562-8576, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34446572

RESUMO

The timing and quality of sleep-wake cycles are regulated by interacting circadian and homeostatic mechanisms. Although the suprachiasmatic nucleus (SCN) is the principal clock, circadian clocks are active across the brain and the respective sleep-regulatory roles of SCN and local clocks are unclear. To determine the specific contribution(s) of the SCN, we used virally mediated genetic complementation, expressing Cryptochrome1 (Cry1) to establish circadian molecular competence in the suprachiasmatic hypothalamus of globally clockless, arrhythmic male Cry1/Cry2-null mice. Under free-running conditions, the rest/activity behavior of Cry1/Cry2-null controls expressing EGFP (SCNCon) was arrhythmic, whereas Cry1-complemented mice (SCNCry1) had coherent circadian behavior, comparable to that of Cry1,2-competent wild types (WTs). In SCNCon mice, sleep-wakefulness, assessed by electroencephalography (EEG)/electromyography (EMG), lacked circadian organization. In SCNCry1 mice, however, it matched WTs, with consolidated vigilance states [wake, rapid eye movement sleep (REMS) and non-REMS (NREMS)] and rhythms in NREMS δ power and expression of REMS within total sleep (TS). Wakefulness in SCNCon mice was more fragmented than in WTs, with more wake-NREMS-wake transitions. This disruption was reversed in SCNCry1 mice. Following sleep deprivation (SD), all mice showed a homeostatic increase in NREMS δ power, although the SCNCon mice had reduced NREMS during the inactive (light) phase of recovery. In contrast, the dynamics of homeostatic responses in the SCNCry1 mice were comparable to WTs. Finally, SCNCon mice exhibited poor sleep-dependent memory but this was corrected in SCNCry1mice. In clockless mice, circadian molecular competence focused solely on the SCN rescued the architecture and consolidation of sleep-wake and sleep-dependent memory, highlighting its dominant role in timing sleep.SIGNIFICANCE STATEMENT The circadian timing system regulates sleep-wake cycles. The hypothalamic suprachiasmatic nucleus (SCN) is the principal circadian clock, but the presence of multiple local brain and peripheral clocks mean the respective roles of SCN and other clocks in regulating sleep are unclear. We therefore used virally mediated genetic complementation to restore molecular circadian functions in the suprachiasmatic hypothalamus, focusing on the SCN, in otherwise genetically clockless, arrhythmic mice. This initiated circadian activity-rest cycles, and circadian sleep-wake cycles, circadian patterning to the intensity of non-rapid eye movement sleep (NREMS) and circadian control of REMS as a proportion of total sleep (TS). Consolidation of sleep-wake established normal dynamics of sleep homeostasis and enhanced sleep-dependent memory. Thus, the suprachiasmatic hypothalamus, alone, can direct circadian regulation of sleep-wake.


Assuntos
Ritmo Circadiano/fisiologia , Criptocromos/biossíntese , Sono/fisiologia , Núcleo Supraquiasmático/metabolismo , Vigília/fisiologia , Animais , Relógios Circadianos/fisiologia , Criptocromos/genética , Eletroencefalografia/métodos , Eletromiografia/métodos , Masculino , Transtornos da Memória , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
3.
J Neurosci ; 41(3): 502-512, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33234609

RESUMO

Circadian (approximately daily) rhythms pervade mammalian behavior. They are generated by cell-autonomous, transcriptional/translational feedback loops (TTFLs), active in all tissues. This distributed clock network is coordinated by the principal circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). Its robust and accurate time-keeping arises from circuit-level interactions that bind its individual cellular clocks into a coherent time-keeper. Cells that express the neuropeptide vasoactive intestinal peptide (VIP) mediate retinal entrainment of the SCN; and in the absence of VIP, or its cognate receptor VPAC2, circadian behavior is compromised because SCN cells cannot synchronize. The contributions to pace-making of other cell types, including VPAC2-expressing target cells of VIP, are, however, not understood. We therefore used intersectional genetics to manipulate the cell-autonomous TTFLs of VPAC2-expressing cells. Measuring circadian behavioral and SCN rhythmicity in these temporally chimeric male mice thus enabled us to determine the contribution of VPAC2-expressing cells (∼35% of SCN cells) to SCN time-keeping. Lengthening of the intrinsic TTFL period of VPAC2 cells by deletion of the CK1εTau allele concomitantly lengthened the period of circadian behavioral rhythms. It also increased the variability of the circadian period of bioluminescent TTFL rhythms in SCN slices recorded ex vivo Abrogation of circadian competence in VPAC2 cells by deletion of Bmal1 severely disrupted circadian behavioral rhythms and compromised TTFL time-keeping in the corresponding SCN slices. Thus, VPAC2-expressing cells are a distinct, functionally powerful subset of the SCN circuit, contributing to computation of ensemble period and maintenance of circadian robustness. These findings extend our understanding of SCN circuit topology.


Assuntos
Comportamento Animal/fisiologia , Ritmo Circadiano/fisiologia , Periodicidade , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/fisiologia , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/fisiologia , Animais , Ritmo Circadiano/genética , Retroalimentação Fisiológica , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Proteínas Mutantes Quiméricas/genética , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Núcleo Supraquiasmático/fisiologia
4.
Nat Commun ; 11(1): 3394, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636383

RESUMO

The hypothalamic suprachiasmatic nuclei (SCN) are the principal mammalian circadian timekeeper, co-ordinating organism-wide daily and seasonal rhythms. To achieve this, cell-autonomous circadian timing by the ~20,000 SCN cells is welded into a tight circuit-wide ensemble oscillation. This creates essential, network-level emergent properties of precise, high-amplitude oscillation with tightly defined ensemble period and phase. Although synchronised, regional cell groups exhibit differentially phased activity, creating stereotypical spatiotemporal circadian waves of cellular activation across the circuit. The cellular circuit pacemaking components that generate these critical emergent properties are unknown. Using intersectional genetics and real-time imaging, we show that SCN cells expressing vasoactive intestinal polypeptide (VIP) or its cognate receptor, VPAC2, are neurochemically and electrophysiologically distinct, but together they control de novo rhythmicity, setting ensemble period and phase with circuit-level spatiotemporal complexity. The VIP/VPAC2 cellular axis is therefore a neurochemically and topologically specific pacemaker hub that determines the emergent properties of the SCN timekeeper.


Assuntos
Ritmo Circadiano , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Núcleo Supraquiasmático/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Relógios Circadianos , Criptocromos/genética , Feminino , Genes Reporter , Teste de Complementação Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Optogenética , Oscilometria , Transdução de Sinais , Núcleo Supraquiasmático/citologia
5.
Eur J Neurosci ; 51(1): 229-240, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30462867

RESUMO

The hypothalamic suprachiasmatic nucleus (SCN) is the principal circadian pacemaker in mammals. Cells in the SCN contain cell-autonomous transcriptional-translational feedback loops, which are synchronised to each other and thereby provide a coherent output to direct synchrony of peripheral clocks located in the brain and body. A major difference between these peripheral clocks and the SCN is the requirement for intercellular coupling mechanisms, which confer robustness, stability and amplitude to the system. There has been remarkable progress to our understanding of the intra- and inter-cellular mechanisms of the SCN circuitry over the last ~20 years, which has come hand-in-hand with the development of new technologies to measure and manipulate the clock.


Assuntos
Relógios Circadianos , Ritmo Circadiano , Animais , Mamíferos , Processamento de Proteína Pós-Traducional , Núcleo Supraquiasmático
6.
Cell ; 177(4): 896-909.e20, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31030999

RESUMO

In mammals, endogenous circadian clocks sense and respond to daily feeding and lighting cues, adjusting internal ∼24 h rhythms to resonate with, and anticipate, external cycles of day and night. The mechanism underlying circadian entrainment to feeding time is critical for understanding why mistimed feeding, as occurs during shift work, disrupts circadian physiology, a state that is associated with increased incidence of chronic diseases such as type 2 (T2) diabetes. We show that feeding-regulated hormones insulin and insulin-like growth factor 1 (IGF-1) reset circadian clocks in vivo and in vitro by induction of PERIOD proteins, and mistimed insulin signaling disrupts circadian organization of mouse behavior and clock gene expression. Insulin and IGF-1 receptor signaling is sufficient to determine essential circadian parameters, principally via increased PERIOD protein synthesis. This requires coincident mechanistic target of rapamycin (mTOR) activation, increased phosphoinositide signaling, and microRNA downregulation. Besides its well-known homeostatic functions, we propose insulin and IGF-1 are primary signals of feeding time to cellular clocks throughout the body.


Assuntos
Relógios Circadianos/fisiologia , Comportamento Alimentar/fisiologia , Proteínas Circadianas Period/metabolismo , Animais , Ritmo Circadiano/fisiologia , Feminino , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais
7.
Biology (Basel) ; 8(1)2019 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-30862123

RESUMO

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.

8.
Science ; 363(6423): 187-192, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30630934

RESUMO

Circadian (~24-hour) rhythms depend on intracellular transcription-translation negative feedback loops (TTFLs). How these self-sustained cellular clocks achieve multicellular integration and thereby direct daily rhythms of behavior in animals is largely obscure. The suprachiasmatic nucleus (SCN) is the fulcrum of this pathway from gene to cell to circuit to behavior in mammals. We describe cell type-specific, functionally distinct TTFLs in neurons and astrocytes of the SCN and show that, in the absence of other cellular clocks, the cell-autonomous astrocytic TTFL alone can drive molecular oscillations in the SCN and circadian behavior in mice. Astrocytic clocks achieve this by reinstating clock gene expression and circadian function of SCN neurons via glutamatergic signals. Our results demonstrate that astrocytes can autonomously initiate and sustain complex mammalian behavior.


Assuntos
Astrócitos/fisiologia , Relógios Circadianos , Ritmo Circadiano , Núcleo Supraquiasmático/fisiologia , Animais , Criptocromos/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia
9.
Proc Natl Acad Sci U S A ; 115(52): E12388-E12397, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30487216

RESUMO

The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional-translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.


Assuntos
Transtornos Cronobiológicos/genética , Criptocromos/genética , Criptocromos/metabolismo , Animais , Transtornos Cronobiológicos/fisiopatologia , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Circadianas Period/metabolismo , Biossíntese de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional , Núcleo Supraquiasmático/metabolismo , Fatores de Transcrição/metabolismo
10.
Nat Rev Neurosci ; 19(8): 453-469, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29934559

RESUMO

The suprachiasmatic nucleus (SCN) of the hypothalamus is remarkable. Despite numbering only about 10,000 neurons on each side of the third ventricle, the SCN is our principal circadian clock, directing the daily cycles of behaviour and physiology that set the tempo of our lives. When this nucleus is isolated in organotypic culture, its autonomous timing mechanism can persist indefinitely, with precision and robustness. The discovery of the cell-autonomous transcriptional and post-translational feedback loops that drive circadian activity in the SCN provided a powerful exemplar of the genetic specification of complex mammalian behaviours. However, the analysis of circadian time-keeping is moving beyond single cells. Technical and conceptual advances, including intersectional genetics, multidimensional imaging and network theory, are beginning to uncover the circuit-level mechanisms and emergent properties that make the SCN a uniquely precise and robust clock. However, much remains unknown about the SCN, not least the intrinsic properties of SCN neurons, its circuit topology and the neuronal computations that these circuits support. Moreover, the convention that the SCN is a neuronal clock has been overturned by the discovery that astrocytes are an integral part of the timepiece. As a test bed for examining the relationships between genes, cells and circuits in sculpting complex behaviours, the SCN continues to offer powerful lessons and opportunities for contemporary neuroscience.


Assuntos
Ritmo Circadiano , Neurônios/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Astrócitos/fisiologia , Relógios Circadianos , Humanos , Transdução de Sinais
11.
FASEB J ; 32(8): 4302-4314, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29561690

RESUMO

Cryptochromes 1 and 2 (CRY1/2) are key components of the negative limb of the mammalian circadian clock. Like many peripheral tissues, Cry1 and -2 are expressed in the retina, where they are thought to play a role in regulating rhythmic physiology. However, studies differ in consensus as to their localization and function, and CRY1 immunostaining has not been convincingly demonstrated in the retina. Here we describe the expression and function of CRY1 and -2 in the mouse retina in both sexes. Unexpectedly, we show that CRY1 is expressed throughout all retinal layers, whereas CRY2 is restricted to the photoreceptor layer. Retinal period 2::luciferase recordings from CRY1-deficient mice show reduced clock robustness and stability, while those from CRY2-deficient mice show normal, albeit long-period, rhythms. In functional studies, we then investigated well-defined rhythms in retinal physiology. Rhythms in the photopic electroretinogram, contrast sensitivity, and pupillary light response were all severely attenuated or abolished in CRY1-deficient mice. In contrast, these physiological rhythms are largely unaffected in mice lacking CRY2, and only photopic electroretinogram rhythms are affected. Together, our data suggest that CRY1 is an essential component of the mammalian retinal clock, whereas CRY2 has a more limited role.-Wong, J. C. Y., Smyllie, N. J., Banks, G. T., Pothecary, C. A., Barnard, A. R., Maywood, E. S., Jagannath, A., Hughes, S., van der Horst, G. T. J., MacLaren, R. E., Hankins, M. W., Hastings, M. H., Nolan, P. M., Foster, R. G., Peirson, S. N. Differential roles for cryptochromes in the mammalian retinal clock.


Assuntos
Criptocromos/metabolismo , Mamíferos/metabolismo , Mamíferos/fisiologia , Retina/metabolismo , Retina/fisiologia , Animais , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Eletrorretinografia/métodos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/fisiologia
12.
Neuron ; 93(6): 1420-1435.e5, 2017 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-28285822

RESUMO

The suprachiasmatic nucleus (SCN) of the hypothalamus orchestrates daily rhythms of physiology and behavior in mammals. Its circadian (∼24 hr) oscillations of gene expression and electrical activity are generated intrinsically and can persist indefinitely in temporal isolation. This robust and resilient timekeeping is generally regarded as a product of the intrinsic connectivity of its neurons. Here we show that neurons constitute only one "half" of the SCN clock, the one metabolically active during circadian daytime. In contrast, SCN astrocytes are active during circadian nighttime, when they suppress the activity of SCN neurons by regulating extracellular glutamate levels. This glutamatergic gliotransmission is sensed by neurons of the dorsal SCN via specific pre-synaptic NMDA receptor assemblies containing NR2C subunits. Remarkably, somatic genetic re-programming of intracellular clocks in SCN astrocytes was capable of remodeling circadian behavioral rhythms in adult mice. Thus, SCN circuit-level timekeeping arises from interdependent and mutually supportive astrocytic-neuronal signaling.


Assuntos
Astrócitos/fisiologia , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Ácido Glutâmico/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora/fisiologia , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/genética
13.
Nat Chem Biol ; 12(10): 776-778, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27571478

RESUMO

Site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion with pyrrolysyl tRNA synthetase (PylRS) and tRNA(Pyl)CUA pairs (and their evolved derivatives) from Methanosarcina sp., forms the basis of powerful approaches to probe and control protein function in cells and invertebrate organisms. Here we demonstrate that adeno-associated viral delivery of these pairs enables efficient genetic code expansion in primary neuronal culture, organotypic brain slices and the brains of live mice.


Assuntos
Aminoácidos/química , Aminoácidos/genética , Aminoacil-tRNA Sintetases/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Código Genético/genética , RNA de Transferência/genética , Aminoácidos/metabolismo , Animais , Dependovirus/genética , Methanosarcina/genética , Camundongos , Estrutura Molecular , RNA de Transferência/metabolismo
14.
Curr Biol ; 26(14): 1880-6, 2016 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-27374340

RESUMO

Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5-7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock.


Assuntos
Relógios Circadianos/genética , Camundongos/genética , Proteínas Circadianas Period/genética , Núcleo Supraquiasmático/metabolismo , Animais , Proteínas Circadianas Period/metabolismo
15.
Proc Natl Acad Sci U S A ; 113(13): 3657-62, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26966234

RESUMO

The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside non-Drd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells.


Assuntos
Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Camundongos , Camundongos Transgênicos , Atividade Motora/genética , Atividade Motora/fisiologia , Plasticidade Neuronal , Neurônios/fisiologia , Fotoperíodo , Receptores de Dopamina D1/deficiência , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/fisiologia , Transdução de Sinais , Núcleo Supraquiasmático/citologia
16.
Proc Natl Acad Sci U S A ; 113(10): 2756-61, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26903623

RESUMO

The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1-CLOCK complexes is suppressed by PER-CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2(Edo/Edo) mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2(Edo) complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2(Edo/Edo); Csnk1e(Tau/Tau) mice and the SCN. These periods are unprecedented in mice. Thus, Per2(Edo) reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.


Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Mutação de Sentido Incorreto , Proteínas Circadianas Period/genética , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Caseína Quinase Iépsilon/genética , Caseína Quinase Iépsilon/metabolismo , Chlorocebus aethiops , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Atividade Motora/genética , Atividade Motora/fisiologia , Proteínas Circadianas Period/química , Proteínas Circadianas Period/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiopatologia
17.
Proc Natl Acad Sci U S A ; 113(10): 2732-7, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26903624

RESUMO

Circadian rhythms in mammals are coordinated by the suprachiasmatic nucleus (SCN). SCN neurons define circadian time using transcriptional/posttranslational feedback loops (TTFL) in which expression of Cryptochrome (Cry) and Period (Per) genes is inhibited by their protein products. Loss of Cry1 and Cry2 stops the SCN clock, whereas individual deletions accelerate and decelerate it, respectively. At the circuit level, neuronal interactions synchronize cellular TTFLs, creating a spatiotemporal wave of gene expression across the SCN that is lost in Cry1/2-deficient SCN. To interrogate the properties of CRY proteins required for circadian function, we expressed CRY in SCN of Cry-deficient mice using adeno-associated virus (AAV). Expression of CRY1::EGFP or CRY2::EGFP under a minimal Cry1 promoter was circadian and rapidly induced PER2-dependent bioluminescence rhythms in previously arrhythmic Cry1/2-deficient SCN, with periods appropriate to each isoform. CRY1::EGFP appropriately lengthened the behavioral period in Cry1-deficient mice. Thus, determination of specific circadian periods reflects properties of the respective proteins, independently of their phase of expression. Phase of CRY1::EGFP expression was critical, however, because constitutive or phase-delayed promoters failed to sustain coherent rhythms. At the circuit level, CRY1::EGFP induced the spatiotemporal wave of PER2 expression in Cry1/2-deficient SCN. This was dependent on the neuropeptide arginine vasopressin (AVP) because it was prevented by pharmacological blockade of AVP receptors. Thus, our genetic complementation assay reveals acute, protein-specific induction of cell-autonomous and network-level circadian rhythmicity in SCN never previously exposed to CRY. Specifically, Cry expression must be circadian and appropriately phased to support rhythms, and AVP receptor signaling is required to impose circuit-level circadian function.


Assuntos
Criptocromos/metabolismo , Receptores de Vasopressinas/metabolismo , Transdução de Sinais , Núcleo Supraquiasmático/metabolismo , Animais , Arritmias Cardíacas/fisiopatologia , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Criptocromos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Núcleo Supraquiasmático/fisiopatologia , Fatores de Tempo
18.
Cell ; 162(3): 607-21, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26232227

RESUMO

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.


Assuntos
Ritmo Circadiano , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Neuropeptídeos/genética , Núcleo Supraquiasmático/metabolismo , Sequência de Aminoácidos , Animais , Regulação para Baixo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Alinhamento de Sequência , Transcrição Genética
19.
Curr Biol ; 24(23): 2838-44, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25454592

RESUMO

Circadian clocks allow anticipation of daily environmental changes. The suprachiasmatic nucleus (SCN) houses the master clock, but clocks are also widely expressed elsewhere in the body. Although some peripheral clocks have established roles, it is unclear what local brain clocks do. We tested the contribution of one putative local clock in mouse histaminergic neurons in the tuberomamillary nucleus to the regulation of the sleep-wake cycle. Histaminergic neurons are silent during sleep, and start firing after wake onset; the released histamine, made by the enzyme histidine decarboxylase (HDC), enhances wakefulness. We found that hdc gene expression varies with time of day. Selectively deleting the Bmal1 (also known as Arntl or Mop3) clock gene from histaminergic cells removes this variation, producing higher HDC expression and brain histamine levels during the day. The consequences include more fragmented sleep, prolonged wake at night, shallower sleep depth (lower nonrapid eye movement [NREM] δ power), increased NREM-to-REM transitions, hindered recovery sleep after sleep deprivation, and impaired memory. Removing BMAL1 from histaminergic neurons does not, however, affect circadian rhythms. We propose that for mammals with polyphasic/nonwake consolidating sleep, the local BMAL1-dependent clock directs appropriately timed declines and increases in histamine biosynthesis to produce an appropriate balance of wake and sleep within the overall daily cycle of rest and activity specified by the SCN.


Assuntos
Fatores de Transcrição ARNTL/fisiologia , Neurônios/metabolismo , Sono/fisiologia , Animais , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica , Histamina/metabolismo , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Privação do Sono , Núcleo Supraquiasmático/fisiologia
20.
J Biol Rhythms ; 29(4): 288-98, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25238857

RESUMO

Within the suprachiasmatic nucleus (SCN) of the hypothalamus, circadian timekeeping and resetting have been shown to be largely dependent on both membrane depolarization and intracellular second-messenger signaling. In both of these processes, voltage-gated calcium channels (VGCCs) mediate voltage-dependent calcium influx, which propagates neural impulses by stimulating vesicle fusion and instigates intracellular pathways resulting in clock gene expression. Through the cumulative actions of these processes, the phase of the internal clock is modified to match the light cycle of the external environment. To parse out the distinct roles of the L-type VGCCs, we analyzed mice deficient in Cav1.2 (Cacna1c) in brain tissue. We found that mice deficient in the Cav1.2 channel exhibited a significant reduction in their ability to phase-advance circadian behavior when subjected to a light pulse in the late night. Furthermore, the study revealed that the expression of Cav1.2 mRNA was rhythmic (peaking during the late night) and was regulated by the circadian clock component REV-ERBα. Finally, the induction of clock genes in both the early and late subjective night was affected by the loss of Cav1.2, with reductions in Per2 and Per1 in the early and late night, respectively. In sum, these results reveal a role of the L-type VGCC Cav1.2 in mediating both clock gene expression and phase advances in response to a light pulse in the late night.


Assuntos
Canais de Cálcio Tipo L/genética , Relógios Circadianos/genética , Ritmo Circadiano/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Animais , Cálcio/metabolismo , Expressão Gênica/genética , Luz , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Circadianas Period/genética , Fotoperíodo , RNA Mensageiro/genética , Núcleo Supraquiasmático/fisiologia
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