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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.
EMBO J ; 40(20): e108614, 2021 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-34487375

RESUMO

Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN.

3.
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
4.
Neuron ; 109(4): 576-596, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33385325

RESUMO

Astrocytes are a large and diverse population of morphologically complex cells that exist throughout nervous systems of multiple species. Progress over the last two decades has shown that astrocytes mediate developmental, physiological, and pathological processes. However, a long-standing open question is how astrocytes regulate neural circuits in ways that are behaviorally consequential. In this regard, we summarize recent studies using Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, and Mus musculus. The data reveal diverse astrocyte mechanisms operating in seconds or much longer timescales within neural circuits and shaping multiple behavioral outputs. We also refer to human diseases that have a known primary astrocytic basis. We suggest that including astrocytes in mechanistic, theoretical, and computational studies of neural circuits provides new perspectives to understand behavior, its regulation, and its disease-related manifestations.


Assuntos
Astrócitos/metabolismo , Transtornos Mentais/metabolismo , Rede Nervosa/metabolismo , Neurônios/metabolismo , Animais , Astrócitos/patologia , Caenorhabditis elegans , Drosophila , Humanos , Transtornos Mentais/genética , Transtornos Mentais/patologia , Camundongos , Rede Nervosa/patologia , Neurônios/patologia , Especificidade da Espécie , Peixe-Zebra
5.
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
6.
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
7.
PLoS Genet ; 16(4): e1008729, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32352975

RESUMO

Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.


Assuntos
Fatores de Transcrição ARNTL/genética , Envelhecimento/genética , Ritmo Circadiano , Fatores de Transcrição ARNTL/metabolismo , Envelhecimento/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Encéfalo/embriologia , Células Cultivadas , Retroalimentação Fisiológica , Fígado/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Músculo Esquelético/metabolismo , Biossíntese de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Célula Única/métodos
8.
J Mol Biol ; 432(12): 3639-3660, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996314

RESUMO

Circadian (approximately daily) rhythms of physiology and behaviour adapt organisms to the alternating environments of day and night. The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian timekeeper of mammals. The mammalian cell-autonomous circadian clock is built around a self-sustaining transcriptional-translational negative feedback loop (TTFL) in which the negative regulators Per and Cry suppress their own expression, which is driven by the positive regulators Clock and Bmal1. Importantly, such TTFL-based clocks are present in all major tissues across the organism, and the SCN is their central co-ordinator. First, we analyse SCN timekeeping at the cell-autonomous and the circuit-based levels of organisation. We consider how molecular-genetic manipulations have been used to probe cell-autonomous timing in the SCN, identifying the integral components of the clock. Second, we consider new approaches that enable real-time monitoring of the activity of these clock components and clock-driven cellular outputs. Finally, we review how intersectional genetic manipulations of the cell-autonomous clockwork can be used to determine how SCN cells interact to generate an ensemble circadian signal. Critically, it is these network-level interactions that confer on the SCN its emergent properties of robustness, light-entrained phase and precision- properties that are essential for its role as the central co-ordinator. Remaining gaps in knowledge include an understanding of how the TTFL proteins behave individually and in complexes: whether particular SCN neuronal populations act as pacemakers, and if so, by which signalling mechanisms, and finally the nature of the recently discovered role of astrocytes within the SCN network.


Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Neurônios/metabolismo , Núcleo Supraquiasmático/metabolismo , Animais , Astrócitos/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/genética , Transdução de Sinais/genética
9.
Cell Metab ; 30(2): 238-250, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31390550

RESUMO

The importance of circadian biology has rarely been considered in pre-clinical studies, and even more when translating to the bedside. Circadian biology is becoming a critical factor for improving drug efficacy and diminishing drug toxicity. Indeed, there is emerging evidence showing that some drugs are more effective at nighttime than daytime, whereas for others it is the opposite. This suggests that the biology of the target cell will determine how an organ will respond to a drug at a specific time of the day, thus modulating pharmacodynamics. Thus, it is now time that circadian factors become an integral part of translational research.


Assuntos
Relógios Circadianos , Ritmo Circadiano/efeitos dos fármacos , Animais , Humanos
10.
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.

11.
Nat Commun ; 10(1): 542, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30710088

RESUMO

The suprachiasmatic nucleus (SCN) co-ordinates circadian behaviour and physiology in mammals. Its cell-autonomous circadian oscillations pivot around a well characterised transcriptional/translational feedback loop (TTFL), whilst the SCN circuit as a whole is synchronised to solar time by its retinorecipient cells that express and release vasoactive intestinal peptide (VIP). The cell-autonomous and circuit-level mechanisms whereby VIP synchronises the SCN are poorly understood. We show that SCN slices in organotypic culture demonstrate rapid and sustained circuit-level circadian responses to VIP that are mediated at a cell-autonomous level. This is accompanied by changes across a broad transcriptional network and by significant VIP-directed plasticity in the internal phasing of the cell-autonomous TTFL. Signalling via ERK1/2 and tuning by its negative regulator DUSP4 are critical elements of the VIP-directed circadian re-programming. In summary, we provide detailed mechanistic insight into VIP signal transduction in the SCN at the level of genes, cells and neural circuit.


Assuntos
Relógios Circadianos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Tirosina Fosfatases/metabolismo , Núcleo Supraquiasmático/fisiologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Sistemas CRISPR-Cas , Relógios Circadianos/genética , Relógios Circadianos/efeitos da radiação , AMP Cíclico/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos da radiação , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos da radiação , Luz , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Camundongos Knockout , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Elementos de Resposta/genética , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/efeitos da radiação , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/efeitos da radiação
12.
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
13.
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
14.
Curr Biol ; 28(15): R816-R822, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30086310

RESUMO

Like it or not, your two suprachiasmatic nuclei (SCN) govern your life: from when you wake up and fall asleep, to when you feel hungry or can best concentrate. Each is composed of approximately 10,000 tightly interconnected neurons, and the pair sit astride the mid-line third ventricle of the hypothalamus, immediately dorsal to the optic chiasm (Figure 1A). Together, they constitute the master circadian clock of the mammalian brain. They generate an internal representation of solar time that is conveyed to every cell in our body and in this way they co-ordinate the daily cycles of physiology and behaviour that adapt us to the twenty-four hour world. The temporary discomfort associated with jetlag is a reminder of the importance of this daily programme, but there is growing recognition that its chronic disruption carries a cost for health of far greater scale. In this primer, we shall briefly review the historical identification of the SCN as the master circadian clock, and then discuss it on three different levels: the cell-autonomous SCN, the SCN as a cellular network and, finally, the SCN as circadian orchestrator. We shall focus on the intrinsic electrical and transcriptional properties of the SCN and how these properties are thought to form an input to, and an output from, its intrinsic cellular clockwork. Second, we shall describe the anatomical arrangement of the SCN, how its sub-regions are delineated by different neuropeptides, and how SCN neurons communicate with each other via these neuropeptides and the neurotransmitter γ-aminobutyric acid (GABA). Finally, we shall discuss how the SCN functions as a circadian oscillator that dictates behaviour, and how intersectional genetic approaches are being used to try to unravel the specific contributions to pacemaking of specific SCN cell populations.


Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Humanos
15.
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
16.
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
17.
Nat Biotechnol ; 36(2): 156-159, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29251727

RESUMO

We develop an approach to tag proteomes synthesized by specific cell types in dissociated cortex, brain slices, and the brains of live mice. By viral-mediated expression of an orthogonal pyrrolysyl-tRNA synthetase-tRNAXXX pair in a cell type of interest and providing a non-canonical amino acid with a chemical handle, we selectively label neuronal or glial proteomes. The method enables the identification of proteins from spatially and genetically defined regions of the brain.


Assuntos
Aminoacil-tRNA Sintetases/genética , Encéfalo/metabolismo , Neurônios/metabolismo , Proteoma/genética , Aminoácidos , Animais , Regulação Enzimológica da Expressão Gênica/genética , Camundongos , Neuroglia/metabolismo , RNA de Transferência/genética
18.
J Biol Rhythms ; 32(5): 380-393, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29098954

RESUMO

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding "big data" that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.


Assuntos
Ritmo Circadiano/genética , Genoma , Genômica , Estatística como Assunto/métodos , Bioestatística , Biologia Computacional/métodos , Genômica/estatística & dados numéricos , Humanos , Metabolômica , Proteômica , Software , Biologia de Sistemas
19.
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
20.
Artigo em Inglês | MEDLINE | ID: mdl-28049647

RESUMO

The suprachiasmatic nucleus (SCN) is the principal circadian clock of the brain, directing daily cycles of behavior and physiology. SCN neurons contain a cell-autonomous transcription-based clockwork but, in turn, circuit-level interactions synchronize the 20,000 or so SCN neurons into a robust and coherent daily timer. Synchronization requires neuropeptide signaling, regulated by a reciprocal interdependence between the molecular clockwork and rhythmic electrical activity, which in turn depends on a daytime Na+ drive and nighttime K+ drag. Recent studies exploiting intersectional genetics have started to identify the pacemaking roles of particular neuronal groups in the SCN. They support the idea that timekeeping involves nonlinear and hierarchical computations that create and incorporate timing information through the interactions between key groups of neurons within the SCN circuit. The field is now poised to elucidate these computations, their underlying cellular mechanisms, and how the SCN clock interacts with subordinate circadian clocks across the brain to determine the timing and efficiency of the sleep-wake cycle, and how perturbations of this coherence contribute to neurological and psychiatric illness.


Assuntos
Relógios Circadianos , Núcleo Supraquiasmático/fisiologia , Animais , Humanos , Neurônios/fisiologia , Núcleo Supraquiasmático/citologia
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