The mammalian suprachiasmatic nucleus (SCN) forms not only the master circadian clock but also a seasonal clock. This neural network of ∼10,000 circadian oscillators encodes season-dependent day-length changes through a largely unknown mechanism. We show that region-intrinsic changes in the SCN fine-tune the degree of network synchrony and reorganize the phase relationship among circadian oscillators to represent day length. We measure oscillations of the clock gene Bmal1, at single-cell and regional levels in cultured SCN explanted from animals raised under short or long days. Coupling estimation using the Kuramoto framework reveals that the network has couplings that can be both phase-attractive (synchronizing) and -repulsive (desynchronizing). The phase gap between the dorsal and ventral regions increases and the overall period of the SCN shortens with longer day length. We find that one of the underlying physiological mechanisms is the modulation of the intracellular chloride concentration, which can adjust the strength and polarity of the ionotropic GABA A -mediated synaptic input. We show that increasing daylength changes the pattern of chloride transporter expression, yielding more excitatory GABA synaptic input, and that blocking GABA A signaling or the chloride transporter disrupts the unique phase and period organization induced by the day length. We test the consequences of this tunable GABA coupling in the context of excitation-inhibition balance through detailed realistic modeling. These results indicate that the network encoding of seasonal time is controlled by modulation of intracellular chloride, which determines the phase relationship among and period difference between the dorsal and ventral SCN.day-length encoding | repulsive coupling | SCN | GABA | chloride T he physiological and behavioral rhythms of all life on earth are bound to the Earth's rotational cycle of ∼24 h. This fundamental rhythm is also affected by the planet's slanted rotational axis, which causes seasonal variations in the length of the day. How life has adapted to anticipate this yearly rhythm is still debated.The suprachiasmatic nucleus (SCN), the central circadian (∼24 h) pacemaker in mammals, consists of ∼10,000 "clock" neurons. These single-cell clocks maintain endogenous rhythms by autoregulatory feedback loops of genes including period (Per) and brain and muscle Arnt-like 1 (Bmal1) (1). Although it was speculated that seasonal rhythms might be encoded in a single cell (2), single-cell oscillations remain similar regardless of the seasonal time that the SCN tissue encodes (3). Seasonal timing is thus proposed to be encoded in the network of the SCN (4-7) through spatial reorganization of the relative phases of clocks within individual cells (8-10). Subsets of SCN clocks form phase clusters (11) that map approximately to dorsal (shell, D-SCN) and ventral (core, V-SCN) regions of the SCN. When measured through a luciferase reporter monitoring oscillations in the Bmal1 gene, the D-SCN and V-SCN clusters show a phase gap, ...
The suprachiasmatic nuclei (SCN), the central circadian pacemakers in mammals, comprise a multiscale neuronal system that times daily events. We use recent advances in graphics processing unit computing to generate a multiscale model for the SCN that resolves cellular electrical activity down to the timescale of individual action potentials and the intracellular molecular events that generate circadian rhythms. We use the model to study the role of the neurotransmitter GABA in synchronizing circadian rhythms among individual SCN neurons, a topic of much debate in the circadian community. The model predicts that GABA signaling has two components: phasic (fast) and tonic (slow). Phasic GABA postsynaptic currents are released after action potentials, and can both increase or decrease firing rate, depending on their timing in the interspike interval, a modeling hypothesis we experimentally validate; this allows flexibility in the timing of circadian output signals. Phasic GABA, however, does not significantly affect molecular timekeeping. The tonic GABA signal is released when cells become very excited and depolarized; it changes the excitability of neurons in the network, can shift molecular rhythms, and affects SCN synchrony. We measure which neurons are excited or inhibited by GABA across the day and find GABA-excited neurons are synchronized by-and GABA-inhibited neurons repelled from-this tonic GABA signal, which modulates the synchrony in the SCN provided by other signaling molecules. Our mathematical model also provides an important tool for circadian research, and a model computational system for the many multiscale projects currently studying brain function.
Mammalian circadian clocks have a hierarchical organization, governed by the suprachiasmatic nucleus (SCN) in the hypothalamus. The brain itself contains multiple loci that maintain autonomous circadian rhythmicity, but the contribution of the non-SCN clocks to this hierarchy remains unclear. We examine circadian oscillations of clock gene expression in various brain loci and discovered that in mouse, robust, higher amplitude, relatively faster oscillations occur in the choroid plexus (CP) compared to the SCN. Our computational analysis and modeling show that the CP achieves these properties by synchronization of “twist” circadian oscillators via gap-junctional connections. Using an in vitro tissue coculture model and in vivo targeted deletion of the Bmal1 gene to silence the CP circadian clock, we demonstrate that the CP clock adjusts the SCN clock likely via circulation of cerebrospinal fluid, thus finely tuning behavioral circadian rhythms.
Together with computer simulations, we show that either the intercellular coupling does not strongly influence the Bmal1 oscillation or the nature of the coupling is more complex than previously assumed. Furthermore, we have found that the region-specific periods are modulated by the light conditions that an animal is exposed to. Based on these, we suggest that the period forms the basis of seasonal coding in the SCN.
Metabolic and cardiovascular processes controlled by the hindbrain exhibit 24 h rhythms, but the extent to which the hindbrain possesses endogenous circadian timekeeping is unresolved. Here we provide compelling evidence that genetic, neuronal, and vascular activities of the brainstem's dorsal vagal complex are subject to intrinsic circadian control with a crucial role for the connection between its components in regulating their rhythmic properties. Robust 24 h variation in clock gene expression in vivo and neuronal firing ex vivo were observed in the area postrema (AP) and nucleus of the solitary tract (NTS), together with enhanced nocturnal responsiveness to metabolic cues. Unexpectedly, we also find functional and molecular evidence for increased penetration of blood borne molecules into the NTS at night. Our findings reveal that the hindbrain houses a local network complex of neuronal and nonneuronal autonomous circadian oscillators, with clear implications for understanding local temporal control of physiology in the brainstem.
Two independent studies, one of them using a computational approach, identified CHRONO, a gene shown to modulate the activity of circadian transcription factors and alter circadian behavior in mice.
Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) is the key enzyme in melatonin synthesis regulated by circadian rhythm. To date, our understanding of the oscillatory mechanism of melatonin has been limited to autoregulatory transcriptional and posttranslational regulations of AANAT mRNA. In this study, we identify three proteins from pineal glands that associate with cis-acting elements within species-specific AANAT 3 untranslated regions to mediate mRNA degradation. These proteins include heterogeneous nuclear ribonucleoprotein R (hnRNP R), hnRNP Q, and hnRNP L. Their RNA-destabilizing function was determined by RNA interference and overexpression approaches. Expression patterns of these factors in pineal glands display robust circadian rhythm. The enhanced levels detected after midnight correlate with an abrupt decline in AANAT mRNA level. A mathematical model for the AANAT mRNA profile and its experimental evidence with rat pinealocytes indicates that rhythmic AANAT mRNA degradation mediated by hnRNP R, hnRNP Q, and hnRNP L is a key process in the regulation of its circadian oscillation.Circadian rhythm is a fundamental biological phenomenon in living organisms (10,41,53). To date, efforts to understand the molecular mechanisms of circadian rhythm have focused mainly on transcriptional regulation. A number of studies show that autoregulatory transcriptional-posttranslational feedback loops are crucial for the rhythmic expression of clock-controlled genes (14,30,40,41,46). However, limited data on the posttranscriptional level are available (45). Since mRNA turnover has notable effects on the synthesis of specific proteins and provides the cell with flexibility in achieving rapid changes at the transcript level (9, 35, 50, 52), it is possible that posttranscriptional regulation functions in the rhythmic expression of circadian genes.Recent evidence supports the existence of posttranscriptional mechanisms. In Drosophila, the degradation of Period (per) mRNA modulates its proper circadian fluctuation (49). The accelerated decay of mouse Per1 (mPer1) mRNA in a tau mutant is additionally suggestive of the presence of a posttranscriptional regulatory pathway (32). In transgenic experiments, the differences between the mRNA fluctuations of clock-controlled genes and reporters were tentatively accounted for by variations in their mRNA stability mediated by 3Ј untranslated regions (3ЈUTRs) (22, 51). In computational modeling approaches, mRNA degradation is assumed in the construction of circadian clock models, although its role in rhythm formation is not currently clear (12, 31). Here, we postulate that dynamic mRNA degradation is essential for the formation of circadian rhythms in clock-controlled gene expression, and we support our theory with mathematical modeling and experimental evidence of rat serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) mRNA rhythms.AANAT is a rate-limiting enzyme in the melatonin synthetic pathway that drives the daily rhythm in the leve...
A circadian clock governs most aspects of mammalian behavior. Although its properties are in part genetically determined, altered light-dark environment can change circadian period length through a mechanism requiring de novo DNA methylation. We show here that this mechanism is mediated not via cell-autonomous clock properties, but rather through altered networking within the suprachiasmatic nuclei (SCN), the circadian “master clock”, which is DNA-methylated in region-specific manner. DNA methylation is necessary to temporally reorganize circadian phasing among SCN neurons, which in turn changes the period length of the network as a whole. Interruption of neural communication by inhibiting neuronal firing or by physical cutting suppresses both SCN reorganization and period changes. Mathematical modeling suggests, and experiments confirm, that this SCN reorganization depends upon GABAergic signaling. Our results therefore show that basic circadian clock properties are governed by dynamic interactions among SCN neurons, with neuroadaptations in network function driven by the environment.
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