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In the mammalian suprachiasmatic nucleus (SCN), noisy cellular oscillators communicate within a neuronal network to generate precise system-wide circadian rhythms. Although the intracellular genetic oscillator and intercellular biochemical coupling mechanisms have been examined previously, the network topology driving synchronization of the SCN has not been elucidated. This network has been particularly challenging to probe, due to its oscillatory components and slow coupling timescale. In this work, we investigated the SCN network at a single-cell resolution through a chemically induced desynchronization. We then inferred functional connections in the SCN by applying the maximal information coefficient statistic to bioluminescence reporter data from individual neurons while they resynchronized their circadian cycling. Our results demonstrate that the functional network of circadian cells associated with resynchronization has small-world characteristics, with a node degree distribution that is exponential. We show that hubs of this small-world network are preferentially located in the central SCN, with sparsely connected shells surrounding these cores. Finally, we used two computational models of circadian neurons to validate our predictions of network structure.

The mammalian circadian clock located in the suprachiasmatic nucleus (SCN) produces robust daily rhythms including rest-wake. SCN neurons synthesize and respond to γ-aminobutyric acid (GABA), but its role remains unresolved. We tested the hypothesis that γ2-and δ-subunits of the GABAA receptor in the SCN differ in their regulation of synchrony among circadian cells. We used two approaches: 1) shRNA to knock-down (KD) the expression of either γ2 or δ subunits in the SCN or 2) knock-in mice harboring a point mutation in the M2 domains of the endogenous GABAA γ2 or δ subunits. KD of either γ2 or δ subunits in the SCN increased daytime running and reduced nocturnal running by reducing their circadian amplitude by a third. Similarly, δ subunit knock-in mice showed decreased circadian amplitude, increased duration of daily activity, and decreased total daily activity. Reduction, or mutation of either γ2 or δ subunits halved the synchrony among, and amplitude of, circadian SCN cells as measured by firing rate or expression of the PERIOD2 protein, in vitro. Surprisingly, overexpression of the γ2 subunit rescued these phenotypes following KD or mutation of the δ subunit, and overexpression of the δ subunit rescued deficiencies due to γ2 subunit KD or mutation. We conclude that γ2 and δ GABAA receptor subunits play similar roles in maintaining circadian synchrony in the SCN and amplitude of daily rest-wake rhythms, but that modulation of their relative densities can change the duration and amplitude of daily activities. [ABSTRACT FROM AUTHOR]

Shift work or transmeridian travel can desynchronize the body's circadian rhythms from local light-dark cycles. The mammalian suprachiasmatic nucleus (SCN) generates and entrains daily rhythms in physiology and behavior. Paradoxically, we found that vasoactive intestinal polypeptide (VIP), a neuropeptide implicated in synchrony among SCN cells, can also desynchronize them. The degree and duration of desynchronization among SCN neurons depended on both the phase and the dose of VIP. A model of the SCN consisting of coupled stochastic cells predicted both the phase- and the dose-dependent response to VIP and that the transient phase desynchronization, or "phase tumbling", could arise from intrinsic, stochastic noise in small populations of key molecules (notably, Period mRNA near its daily minimum). The model also predicted that phase tumbling following brief VIP treatment would accelerate entrainment to shifted environmental cycles. We tested this using a prepulse of VIP during the day before a shift in either a light cycle in vivo or a temperature cycle in vitro. Although VIP during the day does not shift circadian rhythms, the VIP pretreatment approximately halved the time required for mice to reentrain to an 8-h shifted light schedule and for SCN cultures to reentrain to a 10-h shifted temperature cycle. We conclude that VIP below 100 nM synchronizes SCN cells and above 100 nM reduces synchrony in the SCN. We show that exploiting these mechanisms that transiently reduce cellular synchrony before a large shift in the schedule of daily environmental cues has the potential to reduce jet lag.

Patients with cirrhosis exhibit features of circadian disruption. Hyperammonaemia has been suggested to impair both homeostatic and circadian sleep regulation. Here, we tested if hyperammonaemia directly disrupts circadian rhythm generation in the central pacemaker, the suprachiasmatic nuclei (SCN) of the hypothalamus. Wheel‐running activity was recorded from mice fed with a hyperammonaemic or normal diet for ~35 days in a 12:12 light–dark (LD) cycle followed by ~15 days in constant darkness (DD). The expression of the clock protein PERIOD2 (PER2) was recorded from SCN explants before, during and after ammonia exposure, ±glutamate receptor antagonists. In LD, hyperammonaemic mice advanced their daily activity onset time by ~1 h (16.8 ± 0.3 vs. 18.1 ± 0.04 h, p =.009) and decreased their total activity, concentrating it during the first half of the night. In DD, hyperammonaemia reduced the amplitude of daily activity (551.5 ± 27.7 vs. 724.9 ± 59 counts, p =.007), with no changes in circadian period. Ammonia (≥0.01 mM) rapidly and significantly reduced PER2 amplitude, and slightly increased circadian period. The decrease in PER2 amplitude correlated with decreased synchrony among circadian cells in the SCN and increased extracellular glutamate, which was rescued by AMPA glutamate receptor antagonists. These data suggest that hyperammonaemia affects circadian regulation of rest‐activity behaviour by increasing extracellular glutamate in the SCN. [ABSTRACT FROM AUTHOR]

Extracting complex interactions (i.e., dynamic topologies) has been an essential, but difficult, step toward understanding large, complex, and diverse systems including biological, financial, and electrical networks. However, reliable and efficient methods for the recovery or estimation of network topology remain a challenge due to the tremendous scale of emerging systems (e.g., brain and social networks) and the inherent nonlinearity within and between individual units. We develop a unified, data-driven approach to efficiently infer connections of networks (ICON). We apply ICON to determine topology of networks of oscillators with different periodicities, degree nodes, coupling functions, and time scales, arising in silico, and in electrochemistry, neuronal networks, and groups of mice. This method enables the formulation of these large-scale, nonlinear estimation problems as a linear inverse problem that can be solved using parallel computing. Working with data from networks, ICON is robust and versatile enough to reliably reveal full and partial resonance among fast chemical oscillators, coherent circadian rhythms among hundreds of cells, and functional connectivity mediating social synchronization of circadian rhythmicity among mice over weeks. [ABSTRACT FROM AUTHOR]

Although the suprachiasmatic nucleus (SCN) has long been considered the master circadian clock in mammals, the topology of the connections that synchronize daily rhythms among SCN cells is not well understood. We combined experimental and computational methods to infer the directed interactions that mediate circadian synchrony between regions of the SCN. We analyzed PERIOD2 (PER2) expression from SCN slices during and after treatment with tetrodotoxin, allowing us to map connections as cells resynchronized their daily cycling following blockade and restoration of cell-cell communication. Using automated analyses, we found that cells in the dorsal SCN stabilized their periods slower than those in the ventral SCN. A phaseamplitude computational model of the SCN revealed that, to reproduce the experimental results: (1) the ventral SCN had to be more densely connected than the dorsal SCN and (2) the ventral SCN needed strong connections to the dorsal SCN. Taken together, these results provide direct evidence that the ventral SCN entrains the dorsal SCN in constant conditions. [ABSTRACT FROM AUTHOR]

Neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, display daily rhythms in electrical activity with more depolarized resting potentials and higher firing rates during the day than at night. Although these daily variations in the electrical properties of SCN neurons are required for circadian rhythms in physiology and behavior, the mechanisms linking changes in neuronal excitability to the molecular clock are not known. Recently, we reported that mice deficient for either Kcna4 (Kv1.4-/-) or Kcnd2 (Kv4.2-/-; but not Kcnd3, Kv4.3-/-), voltage-gated K+ (Kv) channel pore-forming subunits that encode subthreshold, rapidly activating, and inactivating K+ currents (IA), have shortened (0.5 h) circadian periods in SCN firing and in locomotor activity compared with wild-type (WT) mice. In the experiments here, we used a mouse (Per2Luc) line engineered with a bioluminescent reporter construct, PERIOD2::LUCIFERASE (PER2::LUC), replacing the endogenous Per2 locus, to test the hypothesis that the loss of Kv1.4- or Kv4.2-encoded IA channels also modifies circadian rhythms in the expression of the clock protein PERIOD2 (PER2). We found that SCN explants from Kv1.4-/-Per2Luc and Kv4.2-/- Per2Luc, but not Kv4.3-/-Per2Luc, mice have significantly shorter (by approximately 0.5 h) circadian periods in PER2 rhythms, compared with explants from Per2Luc mice, revealing that the membrane properties of SCN neurons feedback to regulate clock (PER2) expression. The combined loss of both Kv1.4- and Kv4.2-encoded IA channels in Kv1.4-/-/Kv4.2-/-Per2Luc SCN explants did not result in any further alterations in PER2 rhythms. Interestingly, however, mice lacking both Kv1.4 and Kv4.2 show a striking (approximately 1.8 h) advance in their daily activity onset in a light cycle compared with WT mice, suggesting additional roles for Kv1.4- and Kv4.2-encoded IA channels in controlling the light-dependent responses of neurons within and/or outside of the SCN to regulate circadian phase of daily activity. [ABSTRACT FROM AUTHOR]

Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K+) channels regulate daily oscillations in the spontaneous firing rates of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K+ conductance(s) driving these daily rhythms in the repetitive firing rates of SCN neurons, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K+ channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1-/-) or Kcnh3 (Kv12.2-/-) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1-/- and Kv12.2-/- than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1-/-, Kv12.2-/-, and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1-/- and Kv12.2-/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, the pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of the nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1-/-, Kv12.2-/-, and Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals. [ABSTRACT FROM AUTHOR]

Neurons in the suprachiasmatic nucleus (SCN) display coordinated circadian changes in electrical activity that are critical for daily rhythms in physiology, metabolism, and behavior. SCN neurons depolarize spontaneously and fire repetitively during the day and hyperpolarize, drastically reducing firing rates, at night. To explore the hypothesis that rapidly activating and inactivating A-type (IA) voltage-gated K+ (Kv) channels, which are also active at subthreshold membrane potentIAls, are critical regulators of the excitability of SCN neurons, we examined locomotor activity and SCN firing in mice lacking Kv1.4 (Kv1.4-/-), Kv4.2 (Kv4.2-/-), or Kv4.3 (Kv4.3-/-), the pore-forming (α) subunits of IA channels. Mice lacking either Kv1.4 or Kv4.2 α subunits have markedly shorter (0.5 h) periods of locomotor activity than wild-type (WT) mice. In vitro extracellular multi-electrode recordings revealed that Kv1.4-/-and Kv4.2-/-SCN neurons display circadIAn rhythms in repetitive firing, but with shorter periods (0.5 h) than WT cells. In contrast, the periods of wheel-running activity in Kv4.3-/- mice and firing in Kv4.3-/- SCN neurons were indistinguishable from WT animals and neurons. Quantitative real-time PCR revealed that the transcripts encoding all three Kv channelα subunits, Kv1.4, Kv4.2, and Kv4.3, are expressed constitutively throughout the day and night in the SCN. Together, these results demonstrate that Kv1.4- and Kv4.2-encoded IA channels regulate the intrinsic excitability of SCN neurons during the day and night and determine the period and amplitude of circadian rhythms in SCN neuron firing and locomotor behavior. [ABSTRACT FROM AUTHOR]

Behavioral and physiological circadian rhythms in mammals are controlled by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN). Recently, circadian oscillations of hormone secretion, clock gene expression, and electrical activity have been demonstrated in explants of other brain regions. This suggests that some extra-SCN brain regions contain a functional, SCN-independent circadian clock, but in vivo evidence for intrinsic pacemaking is still lacking. We developed a novel method to image bioluminescence in vivo from the main olfactory bulbs (OB) of intact and SCN-lesioned (SCNX) Period1::luciferase rats for 2 d inconstant darkness. The OBs expressed circadian rhythms in situ with a reliable twofold increase from day to night, similar to the phase and amplitude of ex vivo rhythms. In vivo cycling persisted for at least 1 month in the absence of the SCN. To assess indirectly in vivo rhythmicity of other brain areas, we measured the phase-dependence of their in vitro rhythms on the time of surgery. Surgery reliably reset the phase of the pineal gland and vascular organ of the lamina terminalis (VOLT) harvested from SCNX rats but had little effect on the phase of the OB. We deduce that the SCN and OB contain self-sustained circadian oscillators, whereas the pineal gland and VOLT are weak oscillators that require input from the SCN to show coordinated circadian rhythms. We conclude that the mammalian brain comprises a diverse set of SCN-dependent and SCNindependent circadian oscillators. [ABSTRACT FROM AUTHOR]

The suprachiasmatic nucleus (SCN) of the hypothalamus has been termed the master circadian pacemaker of mammals. Recent discoveries of damped circadian oscillators in other tissues have led to the hypothesis that the SCN synchronizes and sustains daily rhythms in these tissues. We studied the effects of constant lighting (LL) and of SCN lesions on behavioral rhythmicity and Period 1 (Per1) gene activity in the SCN and olfactory bulb (OB). We found that LL had similar effects on cyclic locomotor and feeding behaviors and Per1 expression in the SCN but had no effect on rhythmic Period 1 expression in the OB. LL lengthened the period of locomotor and SCN rhythms by ∼1.6 hr. After 2 weeks in LL, nearly 35% of rats lost behavioral rhythmicity. Of these, 90% showed no rhythm in Per1-driven expression in their SCN. Returning the animals to constant darkness rapidly restored their daily cycles of running wheel activity and gene expression in the SCN. In contrast, the OB remained rhythmic with no significant change in period, even when cultured from animals that had been behaviorally arrhythmic for 1 month. Similarly, we found that lesions of the SCN abolished circadian rhythms in behavior but not in the OB. Together, these results suggest that LL causes the SCN to lose circadian rhythmicity and its ability to coordinate daily locomotor and feeding rhythms. The SCN, however, is not required to sustain all rhythms because the OB continues to oscillate in vivo when the SCN is arrhythmic or ablated. [ABSTRACT FROM AUTHOR]

Brief photic stimuli at different circadian times induce differential expression of c-Fos in the suprachiasmatic nuclei (SCN). Whether circadian modulation of light-induced c-Fos expression occurs in other visual projection areas is not known. We addressed this question by estimating the immunohistochemical expression of c-Fos induced by 60 min light pulses at three different circadian times. The areas studied were the SCN, the ventral lateral geniculate nucleus, the intergeniculate leaflet, the ventral tegmental area, the superior colliculus and a non-visual control, the paraventricular thalamic nucleus (PVT). Light pulses induced an increase in the number of c-Fos immunoreactive cells in the SCN as a function of the circadian time. Remaining visual structures showed a light-induced increase in c-Fos expression but this was not dependent on the circadian time. The non-visual control area (PVT) did not respond to light pulses. Since no circadian modulation was found in the intergeniculate leaflet, which receives collateral projections from the same retinal ganglion cells that project to the SCN, nor in other primary visual projection areas, the present findings suggest that the circadian modulation of light-induced c-Fos expression in the SCN depends mainly on the functional properties of its intrinsic neurons. [ABSTRACT FROM AUTHOR]

Fetal SCN grafts into intact rats were used as a model to study coupling among circadian oscillators. The phase relation in metabolic activity was analyzed between host SCN and fetal SCN grafted into the third (n = 24) or lateral (n = 18) ventricles. Host and third ventricle SCN grafts showed metabolic activity rhythms with peak values at CT 6, as extrapolated from drinking rhythmicity. In lateral ventricle grafted animals only the host SCN showed rhythmicity, although peak values occurred as indicated by the drinking rhythms at CT 9. The present data suggest asymmetrical communication between host and grafted SCN depending on graft location, which may be related to different types of signal transmission. [ABSTRACT FROM AUTHOR]

Recently, it has been shown that multiple mammalian cell types express daily rhythms in vitro. Although the suprachiasmatic nucleus (SCN) of the hypothalamus is known to regulate a wide range of circadian behaviors, the role for intrinsic rhythmicity in other tissues is unknown. We tested whether the main olfactory bulb (OB) of mice mediates daily changes in olfaction. We found circadian rhythms in cedar oil-induced c-Fos, a protein marker of cellular excitation, in the mitral and granular layers of the OB and in the piriform cortex (PC). These oscillations persisted in constant darkness with a fourfold change in amplitude and a peak ∼4 h after the onset of daily locomotor activity. Electrolytic lesions of the SCN abolished circadian locomotor rhythms, but not odor-induced c-Fos rhythms in the OB or PC. Furthermore, removal of the OB abolished spontaneous circadian cycling of c-Fos in the PC, shortened the free-running period of locomotor rhythms, and accelerated re-entrainment after a 6 h advance and slowed re-entrainment after a 6 h delay in the light schedule. OB ablation or odorant altered the amplitude of c-Fos rhythms in the SCN and ablation of one OB abolished c-Fos rhythms in the ipsilateral PC, but not in the contralateral OB and PC. We conclude that the OB comprises a master circadian pacemaker, which enhances olfactory responsivity each night, drives rhythms in the PC, and interacts with the SCN to coordinate other daily behaviors. [ABSTRACT FROM AUTHOR]

Circadian rhythms in mammals arise from the spatiotemporal synchronization of ~20,000 neuronal clocks in the Suprachiasmatic Nucleus (SCN). While anatomical, molecular, and genetic approaches have revealed diverse cell types and signaling mechanisms, the network wiring that enables SCN cells to communicate and synchronize remains unclear. To overcome the challenges of revealing functional connectivity from fixed tissue, we developed MITE (Mutual Information & Transfer Entropy), an information theory approach that infers directed cell-cell connections with high fidelity. By analyzing 3447 hours of continuously recorded clock gene expression from 9011 cells in 17 mice, we found that the functional connectome of SCN was highly conserved bilaterally and across mice, sparse, and organized into a dorsomedial and a ventrolateral module. While most connections were local, we discovered long-range connections from ventral cells to cells in both the ventral and dorsal SCN. Based on their functional connectivity, SCN cells can be characterized as circadian signal generators, broadcasters, sinks, or bridges. For example, a subset of VIP neurons acts as hubs that generate circadian signals critical to synchronize daily rhythms across the SCN neural network. Simulations of the experimentally inferred SCN networks recapitulated the stereotypical dorsal-to-ventral wave of daily PER2 expression and ability to spontaneously synchronize, revealing that SCN emergent dynamics are sculpted by cell-cell connectivity. We conclude that MITE provides a powerful method to infer functional connectomes, and that the conserved architecture of cell-cell connections mediates circadian synchrony across space and time in the mammalian SCN., Competing Interests: Competing interests The authors declare no competing interests.

Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K + ) channels regulate daily oscillations in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K + conductance(s) driving these daily rhythms in repetitive firing rates, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K + channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1 -/- ) or Kcnh3 (Kv12.2 -/- ) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1 -/- and Kv12.2 -/- , than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1 -/- , Kv12.2 -/- and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1 -/- and Kv12.2 -/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1 -/- , Kv12.2 -/- , Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.