Hierarchically coupled ultradian oscillators generating robust circadian rhythms

Ensembles of mutually coupled ultradian cellular oscillators have been proposed by a number of authors to explain the generation of circadian rhythms in mammals. Most mathematical models using many coupled oscillators predict that the output period should vary as the square root of the number of participating units, thus being inconsistent with the well-established experimental result that ablation of substantial parts of the suprachiasmatic nuclei (SCN), the main circadian pacemaker in mammals, does not eliminate the overt circadian functions, which show no changes in the phases or periods of the rhythms. From these observations, we have developed a theoretical model that exhibits the robustness of the circadian clock to changes in the number of cells in the SCN, and that is readily adaptable to include the successful features of other known models of circadian regulation, such as the phase response curves and light resetting of the phase.     

Spontaneous synchronization of coupled circadian oscillators

In mammals, the circadian pacemaker, which controls daily rhythms, is located in the suprachiasmatic nucleus (SCN). Circadian oscillations are generated in individual SCN neurons by a molecular regulatory network. Cells oscillate with periods ranging from 20 to 28 h, but at the tissue level, SCN neurons display significant synchrony, suggesting a robust intercellular coupling in which neurotransmitters are assumed to play a crucial role. We present a dynamical model for the coupling of a population of circadian oscillators in the SCN. The cellular oscillator, a three-variable model, describes the core negative feedback loop of the circadian clock. The coupling mechanism is incorporated through the global level of neurotransmitter concentration. Global coupling is efficient to synchronize a population of 10,000 cells. Synchronized cells can be entrained by a 24-h light-dark cycle. Simulations of the interaction between two populations representing two regions of the SCN show that the driven population can be phase-leading. Experimentally testable predictions are: 1), phases of individual cells are governed by their intrinsic periods; and 2), efficient synchronization is achieved when the average neurotransmitter concentration would dampen individual oscillators. However, due to the global neurotransmitter oscillation, cells are effectively synchronized.     

Temperature synchronization of the Drosophila circadian clock

BACKGROUND: Circadian clocks are synchronized by both light:dark cycles and by temperature fluctuations. Although it has long been known that temperature cycles can robustly entrain Drosophila locomotor rhythms, nothing is known about the molecular mechanisms involved. RESULTS: We show here that temperature cycles induce synchronized behavioral rhythms and oscillations of the clock proteins PERIOD and TIMELESS in constant light, a situation that normally leads to molecular and behavioral arrhythmicity. We show that expression of the Drosophila clock gene period can be entrained by temperature cycles in cultured body parts and isolated brains. Further, we show that the phospholipase C encoded by the norpA gene contributes to thermal entrainment, suggesting that a receptor-coupled transduction cascade signals temperature changes to the circadian clock. We initiated the further genetic dissection of temperature-entrainment and isolated the novel Drosophila mutation nocte, which is defective in molecular and behavioral entrainment by temperature cycles but synchronizes normally to light:dark cycles. CONCLUSIONS: We conclude that temperature synchronization of the circadian clock is a tissue-autonomous process that is able to override the arrhythmia-inducing effects of constant light. Our data suggest that it involves a cell-autonomous signal-transduction cascade from a thermal receptor to the circadian clock. This process includes the function of phospholipase C and the product specified by the novel mutation nocte.     

The circadian locomotor rhythm of Hemideina thoracica (Orthoptera; Stenopelmatidae): the circadian clock as a population of interacting oscillators

ABSTRACT. The behaviour of the circadian locomotor rhythm of the New Zealand weta, Hemideina thoracica (White), supports the model that the underlying pacemaker consists of a population of weakly coupled oscillators. Certain patterns of locomotor activity, previously demonstrated almost exclusively in vertebrates, are presented here as evidence for the above hypothesis. They include after-effects of various pre-treatments, rhythm-splitting and spontaneous changes in the rhythm. After-effects, which describe the unstable behaviour of free-running circadian rhythms following particular experimental perturbations, have been observed in Hemideina following single light pulses, constant dim light, and laboratory and natural entrainment. Period changes occurred in the activity rhythm after single light pulses of 8-h and 12-h duration (25 lx). Constant dim light (0.1 lx) increased the free-running period (τ) of the activity rhythm, but the after-effect of constant dim light was either an increase or a decrease in τ. After-effects upon both τ and the active phase length of the activity rhythm were found following non-24-h light entrainment cycles with 8-h and 12-h light phases of 25 lx. Qualitative measurements of these after-effects upon τ are presented which reveal a relationship between both the direction and amount of change in τ, and the difference between entrainment cycle length (T) and pre-entrainment free-running period. The after-effect of natural entrainment was an initial short-period free-run (τ < 24h) lasting 5–10 days, generally followed by a rapid period lengthening to τ= 25–26 h. Support for the population model was provided by spontaneous dampening, recovery, and period changes of the rhythm, together with the disruption of the active phase following critical light perturbations, and rhythm-splitting. These Hemideina results are compared with the simulations of the Coupled Stochastic System of Enright (1980).     

Membrane electrical excitability is necessary for the free-running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free-running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell-autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K(+) channel stops the free-running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free-running larval pacemaker clock-like that of the adult pacemaker neurons they give rise to-requires membrane electrical activity to oscillate. In contrast to the free-running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light-dark) conditions. This specific disruption of the free-running clock caused by targeted K(+) channel expression likely reflects a specific cell-autonomous clock-membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology.     

Two coupled neural oscillators as a model of the circadian pacemaker

Pittendrigh first found that the circadian rhythm of locomotor activity in nocturnal rodents split into two components. Hoffman then reported that the splitting phenomenon was even more reproducible in the small diurnal primate Tupaia. These “splitting” experiments and many other experiments suggest that two coupled oscillators may constitute the circadian pacemaker system. Pittendrigh proposed a phenomenological two-oscillator model. Daan and Berde developed a quantitative model assuming that the interaction between the two constituent oscillators is by instantaneous resets. Their model system can simulate several qualitative features in the experimental data. As the assumption of instantaneous resets seems to be unnatural, we study two limit cycle oscillators, which are coupled continuously to each other, as a model of the circadian pacemaker. We assume the following points, (i) One oscillator in a resting state does not affect another oscillator, (ii) Two oscillators are identical, (iii) The coupling is symmetrical. By the theory of Hopf bifurcation it is found that the general two-oscillator system has two stable periodic solutions. One is the in-phase solution where the two constituent oscillators oscillate in phase synchrony. Another is the anti-phase solution where the two oscillators oscillate 180 ° out of phase. The former corresponds to a single pattern of locomotor activity and the latter corresponds to a splitting pattern. Furthermore, we study specific two-neural oscillators, which are linearly coupled to each other. By the method of secondary bifurcation we find that the model shows simultaneous stability of the two alternative phase relationships and the hysteresis phenomena found in Tupaia. A natural period of the uncoupled constituent oscillator is longer than that of the in-phase solution but it is shorter than that of the anti-phase solution. This is in agreement with the data of Tupaia.     

Further evidence that dioscorea latent virus is a potexvirus

Dioscorea latent virus (DLV) was isolated from Dioscoreafloribunda but was not detected in any of 37 cvs of D. alata, D. bulbifera, D. esculenta or D. rotundata from eight countries. It was readily sap-transmitted to 13 of 34 species from five of 12 families; it induced symptomless systemic infection in Nicotiana benthamiana and N. megalosiphon, but only symptomless local infection in other hosts. DLV was stable in vitro: sap from infected N. megalosiphon was still infective after 10 min at 75–80 ^oC but not 85 ^oC, after dilution to 10^-6 but not 10^-7, and after at least 12 months at 23 ^oC. DLV was not transmitted through seed, by contact or by the aphids Aphis gossypii and Myzus persicae. DLV had filamentous particles most of which measured c. 350–900 nm in purified preparations, with two modal lengths of 445 and 875 nm; the particles sedimented as two components with sedimentation coefficients of 111 S and 131 S and had a buoyant density in caesium chloride of 1–33 g cm^-3. The virus had a single nucleic acid species with a mol. wt of 2–3 ± 04 × 10⁶ and usually produced two protein bands (mol. wts 24 900 and 23 100) in SDS-PAGE, although virus preparations made in the presence of chloroform yielded only the larger polypeptide. DLV was serologically distantly related to commelina X and lily X viruses, but not to 11 other established or possible members of the potexvirus group. These results provide further evidence that DLV is a distinct but definitive potexvirus.     

Genome-wide screenings for circadian clock genes in Drosophila

Sleep and Biological Rhythms - A high-density oligonucleotide probe array (GeneChip) has been used to learn how gene expression is globally regulated by the circadian clock mechanism. Here I review...     

Mechanisms of clock output in the Drosophila circadian pacemaker system

Molecular oscillations that underlie the circadian clock are coupled to different output signals by which daily rhythms in downstream events are evoked and/or synchronized. Here the authors review the literature that describes circadian output mechanisms in Drosophila. They begin at the most proximal level, within oscillator cells themselves, by surveying studies of rhythmic gene expression within Drosophila heads. Next the authors describe the several neuron groups that compose the circadian pacemaker network underlying rhythmic locomotor activity, and they detail current models of how that network is organized and coordinated. The authors outline the body of evidence that describes a role for the neuropeptide pigment dispersing factor (PDF) as a circadian transmitter in the fly brain. Finally, in the context of PDF, they consider studies that address mechanisms of signaling from the circadian pacemaker network to downstream neurons and nonneuronal cells that directly control rhythmic outputs.     

Temporal pattern of LH secretion: regulation by multiple ultradian oscillators versus a single circadian oscillator

The possibility that the 24h rhythm output is the composite expression of ultradian oscillators of varying periodicities was examined by assessing the effect of external continuously or pulsed (20-minute) Gonadotropinreleasing hormone (GnRH) infusions on in vitro luteinizing hormone (LH) release patterns from female mouse pituitaries during 38h study spans. Applying stepwise analyses (spectral, cosine fit, best-fit curve, and peak detection analyses) revealed the waveform shape of LH release output patterns over time is composed of several ultradian oscillations of different periods. The results further substantiated previous observations indicating the pituitary functions as an autonomous clock. The GnRH oscillator functions as a pulse generator and amplitude regulator, but it is not the oscillator that drives the ultradian LH release rhythms. At different stages of the estrus cycle, the effect of GnRH on the expression of ultradian periodicities varies, resulting in the modification of their amplitudes but not their periods. The functional output from the system of ultradian oscillators may superimpose a “circadian or infradian phenotype” on the observed secretion pattern. An “amplitude control” hypothesis is proposed: The temporal pattern of LH release is governed by several oscillators that function in conjunction with one another and are regulated by an amplitude-controlled mechanism. Simulated models show that such a mechanism results in better adaptive response to environmental requirements than does a single circadian oscillator. (Chronobiology International, 18(3), 399-412, 2001)     

Spike amplitude of single-unit responses in antennal sensillae is controlled by the Drosophila circadian clock

Circadian changes in membrane potential and spontaneous firing frequency have been observed in microbial systems, invertebrates, and mammals. Oscillators in olfactory sensory neurons (OSNs) from Drosophila are both necessary and sufficient to sustain rhythms in electroanntenogram (EAG) responses, suggesting that odorant receptors (ORs) and/or OR-dependent processes are under clock control. We measured single-unit responses in different antennal sensillae from wild-type, clock mutant, odorant-receptor mutant, and G protein-coupled receptor kinase 2 (Gprk2) mutant flies to examine the cellular and molecular mechanisms that drive rhythms in olfaction. Spontaneous spike amplitude, but not spontaneous or odor-induced firing frequency, is under clock control in ab1 and ab3 basiconic sensillae and T2 trichoid sensillae. Mutants lacking odorant receptors in dendrites display constant low spike amplitudes, and the reduction or increase of levels of GPRK2 in OSNs results in constant low or constant high spontaneous spike amplitudes, respectively. We conclude that spike amplitude is controlled by circadian clocks in basiconic and trichoid sensillae and requires GPRK2 expression and the presence of functional ORs in dendrites. These results argue that rhythms in GPRK2 levels control OR localization and OR-dependent ion channel activity and/or composition to mediate rhythms in spontaneous spike amplitude.     

Short-period mutations of per affect a double-time-dependent step in the Drosophila circadian clock

Circadian (24 hour) PERIOD (PER) protein oscillation is dependent on the double-time (dbt) gene, a casein kinase Ivarepsilon homolog [1-3]. Without dbt activity, hypophosphorylated PER proteins over-accumulate, indicating that dbt is required for PER phosphorylation and turnover [3,4]. There is evidence of a similar role for casein kinase Ivarepsilon in the mammalian circadian clock [5,6]. We have isolated a new dbt allele, dbt(ar), which causes arrhythmic locomotor activity in homozygous viable adults, as well as molecular arrhythmicity, with constitutively high levels of PER proteins, and low levels of TIMELESS (TIM) proteins. Short-period mutations of per, but not of tim, restore rhythmicity to dbt(ar) flies. This suppression is accompanied by a restoration of PER protein oscillations. Our results suggest that short-period per mutations, and mutations of dbt, affect the same molecular step that controls nuclear PER turnover. We conclude that, in wild-type flies, the previously defined PER'short domain' [7,8] may regulate the activity of DBT on PER.     

Further evidence that the CCK2 receptor is coupled to two transduction pathways using site-directed mutagenesis

A heterogeneity of CCK2 receptors has been reported which could correspond to different states of coupling to G proteins and/or association with different second messenger systems. To investigate these hypotheses, the wild-type CCK2 receptor and three mutants F347A, D100N and K333M/K334T/R335L, expected to modify the coupling of the G protein with the third intracellular loop of the receptor, were transfected into Cos-7 cells and their binding and signalling properties were evaluated using the natural ligand CCK8. Activation of wild-type as well as F347A, D100N or K333M/K334T/R335L CCK2 receptors by this ligand led to a similar arachidonic acid release which was blocked by pertussis toxin and the phospholipase A2 inhibitor, mepacrine. Nevertheless, in contrast to the wild-type CCK2 receptor, addition of CCK8 to cells transfected with the F347A or K333M/K334T/R335L mutants did not result in the production of inositol phosphates while the maximum increase in this second messenger formation was reduced by 30% with the D100N mutant. Taken together, these results suggest that the CCK2 receptor is coupled to two G proteins and that Phe347 and the cluster of basic residues K333/K334/R335 probably play a key role in Gq protein stimulation leading to inositol phosphate production but not in activation of the G protein coupled to phospholipase A2. These data bring additional support at the molecular level to the existence of different affinity states of CCK2 receptors suggested from the results of binding assays and behavioural studies.     

The Drosophila dCREB2 gene affects the circadian clock

We report the role of dCREB2, the Drosophila homolog of CREB/CREM, in circadian rhythms. dCREB2 activity cycles with a 24 hr rhythm in flies, both in a light:dark cycle and in constant darkness. A mutation in dCREB2 shortens circadian locomotor rhythm in flies and dampens the oscillation of period, a known clock gene. Cycling dCREB2 activity is abolished in a period mutant, indicating that dCREB2 and Period affect each other and suggesting that the two genes participate in the same regulatory feedback loop. We propose that dCREB2 supports cycling of the Period/Timeless oscillator. These findings support CREB's role in mediating adaptive behavioral responses to a variey of environmental stimuli (stress, growth factors, drug addiction, circadian rhythms, and memory formation) in mammals and long-term memory formation and circadian rhythms in Drosophila.     

Unique self-sustaining circadian oscillators within the brain of Drosophila melanogaster

In Drosophila circadian rhythms persist in constant darkness (DD). The small ventral Lateral Neurons (s-LN_v) mainly control the behavioral circadian rhythm in consortium with the large ventral Lateral Neurons (l-LN_v) and dorsal Lateral Neurons (LN_d). It is believed that the molecular oscillations of clock genes are the source of this persistent behavior. Indeed the s-LN_v, LN_d, Dorsal Neurons (DN)-DN₂ and DN₃ displayed self-sustained molecular oscillations in DD both at RNA and protein levels, except the DN₂ oscillates in anti-phase. In contrast, the l-LN_v and DN₁ displayed self-sustained oscillations at the RNA level, but protein oscillations quickly dampened. Having self-sustained and dampened molecular oscillators together in the DN groups suggested that they play different roles. However, all DN groups seemed to contribute together to the light-dark (LD) behavioral rhythm. The LD entrainment of LN oscillators is achieved through Rhodopsin (RH) and Cryptochrome (CRY). CRY's expression in all DN groups implicates also its role in LD entrainment of DN, like in DN₁. However, mutations in cry and glass that did not inflict LD synchronization of the DN₂, DN₃ oscillator implicate the existence of a novel photoreceptor at least in DN₃.