mPer2 antisense oligonucleotides inhibit mPER2 expression but not circadian rhythms of physiological activity in cultured suprachiasmatic nucleus neurons

Various day-night rhythms, observed at molecular, cellular, and behavioral levels, are governed by an endogenous circadian clock, predominantly functioning in the hypothalamic suprachiasmatic nucleus (SCN). A class of clock genes, mammalian Period (mPer), is known to be rhythmically expressed in SCN neurons, but the correlation between mPER protein levels and autonomous rhythmic activity in SCN neurons is not well understood. Therefore, we blocked mPer translation using antisense phosphothioate oligonucleotides (ODNs) for mPer1 and mPer2 mRNAs and examined the effects on the circadian rhythm of cytosolic Ca2+ concentration and action potentials in SCN slice cultures. Treatment with mPer2 ODNs (20microM for 3 days) but not randomized control ODNs significantly reduced mPER2 immunoreactivity (-63%) in the SCN. Nevertheless, mPer1/2 ODNs treatment inhibited neither action potential firing rhythms nor cytosolic Ca2+ rhythms. These suggest that circadian rhythms in mPER protein levels are not necessarily coupled to autonomous rhythmic activity in SCN neurons.     

Multicellular model for intercellular synchronization in circadian neural networks

We developed a multicellular model characterized by a high degree of heterogeneity to investigate possible mechanisms that underlie circadian network synchronization and rhythmicity in the suprachiasmatic nucleus (SCN). We populated a two-dimensional grid with 400 model neurons coupled via γ-aminobutyric acid (GABA) and vasoactive intestinal polypeptide (VIP) neurotransmitters through a putative Ca²⁺ mediated signaling cascade to investigate their roles in gene expression and electrical firing activity of cell populations. As observed experimentally, our model predicted that GABA would affect the amplitude of circadian oscillations but not synchrony among individual oscillators. Our model recapitulated experimental findings of decreased synchrony and average periods, loss of rhythmicity, and reduced circadian amplitudes as VIP signaling was eliminated. In addition, simulated increases of VIP reduced periodicity and synchrony. We therefore postulated a physiological range of VIP within which the system is able to produce sustained and synchronized oscillations. Our model recapitulated experimental findings of diminished amplitudes and periodicity with decreasing intracellular Ca²⁺ concentrations, suggesting that such behavior could be due to simultaneous decrease of individual oscillation amplitudes and population synchrony. Simulated increases in Cl⁻ levels resulted in increased Cl⁻ influx into the cytosol, a decrease of inhibitory postsynaptic currents, and ultimately a shift of GABA-elicited responses from inhibitory to excitatory. The simultaneous reduction of IPSCs and increase in membrane resting potential produced GABA dose-dependent increases in firing rates across the population, as has been observed experimentally. By integrating circadian gene regulation and electrophysiology with intracellular and intercellular signaling, we were able to develop the first (to our knowledge) multicellular model that allows the effects of clock genes, electrical firing, Ca²⁺, GABA, and VIP on circadian system behavior to be predicted.     

Gastrin-releasing peptide modulates fast delayed rectifier potassium current in Per1-expressing SCN neurons

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) drives and maintains 24-h physiological rhythms, the phases of which are set by the local environmental light-dark cycle. Gastrin-releasing peptide (GRP) communicates photic phase setting signals in the SCN by increasing neurophysiological activity of SCN neurons. Here, the ionic basis for persistent GRP-induced changes in neuronal activity was investigated in SCN slice cultures from Per1::GFP reporter mice during the early night. Recordings from Per1 -fluorescent neurons in SCN slices several hours after GRP treatment revealed a significantly greater action potential frequency, a significant increase in voltage-activated outward current at depolarized potentials, and a significant increase in 4-aminopyridine-sensitive fast delayed rectifier (fDR) potassium currents when compared to vehicle-treated slices. In addition, the persistent increase in spike rate following early-night GRP application was blocked in SCN neurons from mice deficient in Kv3 channel proteins. Because fDR currents are regulated by the clock and are elevated in amplitude during the day, the present results support the model that GRP delays the phase of the clock during the early night by prolonging day-like membrane properties of SCN cells. Furthermore, these findings implicate fDR currents in the ionic basis for GRP-mediated entrainment of the primary mammalian circadian pacemaker.     

LACK OF CALBINDIN-D28K ALTERS RESPONSE OF THE MURINE CIRCADIAN CLOCK TO LIGHT

A strong stimulus adjusting the circadian clock to the prevailing light-dark cycle is light. However, the circadian clock is reset by light only at specific times of the day. The mechanisms mediating such gating of light input to the CNS are not well understood. There is evidence that Ca²⁺ ions play an important role in intracellular signaling mechanisms, including signaling cascades stimulated by light. Therefore, Ca²⁺ is hypothesized to play a role in the light-mediated resetting of the circadian clock. Calbindin-D28k (CB; gene symbol: Calb1) is a Ca²⁺ binding protein implicated in Ca²⁺ homeostasis and sensing. The absence of this protein influences Ca²⁺ buffering capacity of a cell, alters spatio-temporal aspects of intracellular Ca²⁺ signaling, and hence might alter transmission of light information to the circadian clock in neurons of the suprachiasmatic nuclei (SCN). We tested mice lacking a functional Calb1 gene (Calb1^?/?) and found an increased phase-delay response to light applied at circadian time (CT) 14 in these animals. This is accompanied by elevated induction of Per2 gene expression in the SCN. Period length and circadian rhythmicity were comparable between Calb1^?/? and wild-type animals. Our findings indicate an involvement of CB in the signaling pathway that modulates the behavioral and molecular response to light. (Author correspondence: urs.albrecht@unifr.ch)     

The role of Clock in the plasticity of circadian entrainment

The mammalian circadian clock lying in suprachiasmatic nucleus (SCN) is synchronized to about 24 h by the environmental light-dark cycle (LD). The circadian clock exhibits limits of entrainment above and below 24 h, beyond which it will not entrain. Little is known about the mechanisms regulating the limits of entrainment. In this study, we show that wild-type mice entrain to only an LD 24 h cycle, whereas Clock mutant mice can entrain to an LD 24, 28, and 32 h except for LD 20 h and LD 36 h cycle. Under an LD 28 h cycle, Clock mutant mice showed a clear rhythm in Per2 mRNA expression in the SCN and behavior. Light response was also increased. This is the first report to show that the Clock mutation makes it possible to adapt the circadian oscillator to a long period cycle and indicates that the clock gene may have an important role for the limits of entrainment of the SCN to LD cycle.     

Modeling the spontaneous activity in suprachiasmatic nucleus neurons: Role of cation single channels

A population of interconnected neurons of the mammalian suprachiasmatic nuclei (SCN) controls circadian rhythms in physiological functions. In turn, a circadian rhythm of individual neurons is driven by intracellular processes, which via activation of specific membrane channels, produce circadian modulation of electrical firing rate. Yet the membrane target(s) of the cellular clock have remained enigmatic. Previously, subthreshold voltage-dependent cation (SVC) channels have been proposed as the membrane target of the cellular clock responsible for circadian modulation of the firing rate in SCN neurons. We tested this hypothesis with computational modeling based on experimental results from on-cell recording of SVC channel openings in acutely isolated SCN neurons and long-term continuous recording of activity from dispersed SCN neurons in a multielectrode array dish (MED). The model reproduced the circadian behavior if the number of SVC channels or their kinetics were modulated in accordance with protein concentration in a model of the intracellular clock (Scheper et al., 1999. J. Neurosci. 19, 40-47). Such modulation changed the average firing rate of the model neuron from zero (“subjective-night” silence) up to 18 Hz (“subjective-day” peak). Furthermore, the variability of interspike intervals (ISI) and the circadian pattern of firing rate (i.e. silence-to-activity ratio and shape of circadian peaks) are in reasonable agreement with experimental data obtained in dispersed SCN neurons in MED. These results suggest that the variability of ISI in intact SCN neurons is mostly due to stochastic single-channel openings, and that the circadian pattern of the firing rate is specified by threshold properties of dependence of the spontaneous firing rate on the number of single channels (R-N relationship). This plausible mathematical modeling supports the hypothesis that SVC channels could be a critical element in circadian modulation of firing rate in SCN neurons.     

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca²⁺ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.     

Interferon-gamma alters electrical activity and clock gene expression in suprachiasmatic nucleus neurons

The proinflammatory cytokine interferon (IFN-gamma) is an immunomodulatory molecule released by immune cells. It was originally described as an antiviral agent but can also affect functions in the nervous system including circadian activity of the principal mammalian circadian pacemaker, the suprachiasmatic nucleus. IFN-gamma and the synergistically acting cytokine tumor necrosis factor-alpha acutely decrease spontaneous excitatory postsynaptic activity and alter spiking activity in tissue preparations of the SCN. Because IFN-gamma can be released chronically during infections, the authors studied the long-term effects of IFN-gamma on SCN neurons by treating dispersed rat SCN cultures with IFN-gamma over a 4-week period. They analyzed the effect of the treatment on the spontaneous spiking pattern and rhythmic expression of the "clock gene," Period 1. They found that cytokine-treated cells exhibited a lower average spiking frequency and displayed a more irregular firing pattern when compared with controls. Furthermore, long-term treatment with IFN-gamma in cultures obtained from a transgenic Per1-luciferase rat significantly reduced the Per1-luc rhythm amplitude in individual SCN neurons. These results show that IFN-gamma can alter the electrical properties and circadian clock gene expression in SCN neurons. The authors hypothesize that IFN-gamma can modulate circadian output, which may be associated with sleep and rhythm disturbances observed in certain infections and in aging.     

Modeling the electrophysiology of suprachiasmatic nucleus neurons

Neurons in the SCN act as the central circadian (approximately 24-h) pacemaker in mammals. Using measurements of the ionic currents in SCN neurons, the authors fit a Hodgkin-Huxley-type model that accurately reproduces slow (approximately 28 Hz) neural firing as well as the contributions of ionic currents during an action potential. When inputs of other SCN neurons are considered, the model accurately predicts the fractal nature of firing rates and the appearance of random bursting. In agreement with experimental data, the molecular clock within these neurons modulates the firing rate through small changes in the concentration of internal calcium, calcium channels, or potassium channels. Predictions are made on how signals from other neurons can start, stop, speed up, or slow down firing. Only a slow sodium inactivation variable and voltage do not reach equilibrium during the interval between action potentials, and based on this finding, a reduced model is formulated.     

Cell Type-Specific Functions of Period Genes Revealed by Novel Adipocyte and Hepatocyte Circadian Clock Models

In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.     

Knockdown of clock genes in the suprachiasmatic nucleus blocks prolactin surges and alters FRA expression in the locus coeruleus of female rats

The nature of the circadian signal from the suprachiasmatic nucleus (SCN) required for prolactin (PRL) surges is unknown. Because the SCN neuronal circadian rhythm is determined by a feedback loop of Period (Per) 1, Per2, and circadian locomotor output cycles kaput (Clock) gene expressions, we investigated the effect of SCN rhythmicity on PRL surges by disrupting this loop. Because lesion of the locus coeruleus (LC) abolishes PRL surges and these neurons receive SCN projections, we investigated the role of SCN rhythmicity in the LC neuronal circadian rhythm as a possible component of the circadian mechanism regulating PRL surges. Cycling rats on proestrous day and estradiol-treated ovariectomized rats received injections of antisense or random-sequence deoxyoligonucleotide cocktails for clock genes (Per1, Per2, and Clock) in the SCN, and blood samples were taken for PRL measurements. The percentage of tyrosine hydroxylase-positive neurons immunoreactive to Fos-related antigen (FRA) was determined in ovariectomized rats submitted to the cocktail injections and in a 12:12-h light:dark (LD) or constant dark (DD) environment. The antisense cocktail abolished both the proestrous and the estradiol-induced PRL surges observed in the afternoon and the increase of FRA expression in the LC neurons at Zeitgeber time 14 in LD and at circadian time 14 in DD. Because SCN afferents and efferents were probably preserved, the SCN rhythmicity is essential for the magnitude of daily PRL surges in female rats as well as for LC neuronal circadian rhythm. SCN neurons therefore determine PRL secretory surges, possibly by modulating LC circadian neuronal activity.     

Modeling the Cellular Mechanisms and Olfactory Input Underlying the Triphasic Response of Moth Pheromone-Sensitive Projection Neurons

In the antennal lobe of the noctuid moth Agrotis ipsilon, most pheromone-sensitive projection neurons (PNs) exhibit a triphasic firing pattern of excitation (E₁)-inhibition (I)-excitation (E₂) in response to a pulse of the sex pheromone. To understand the mechanisms underlying this stereotypical discharge, we developed a biophysical model of a PN receiving inputs from olfactory receptor neurons (ORNs) via nicotinic cholinergic synapses. The ORN is modeled as an inhomogeneous Poisson process whose firing rate is a function of time and is fitted to extracellular data recorded in response to pheromone stimulations at various concentrations and durations. The PN model is based on the Hodgkin-Huxley formalism with realistic ionic currents whose parameters were derived from previous studies. Simulations revealed that the inhibitory phase I can be produced by a SK current (Ca²⁺-gated small conductance K⁺ current) and that the excitatory phase E₂ can result from the long-lasting response of the ORNs. Parameter analysis further revealed that the ending time of E₁ depends on some parameters of SK, Ca²⁺, nACh and Na⁺ currents; I duration mainly depends on the time constant of intracellular Ca²⁺ dynamics, conductance of Ca²⁺ currents and some parameters of nACh currents; The mean firing frequency of E₁ and E₂ depends differentially on the interaction of various currents. Thus it is likely that the interplay between PN intrinsic currents and feedforward synaptic currents are sufficient to generate the triphasic firing patterns observed in the noctuid moth A. ipsilon.     

Acid sensing ion channels regulate neuronal excitability by inhibiting BK potassium channels

Acid sensing ion channels (ASICs), Ca²⁺ and voltage-activated potassium channels (BK) are widely present throughout the central nervous system. Previous studies have shown that when expressed together in heterologous cells, ASICs inhibit BK channels, and this inhibition is relieved by acidic extracellular pH. We hypothesized that ASIC and BK channels might interact in neurons, and that ASICs may regulate BK channel activity. We found that ASICs inhibited BK currents in cultured wild-type cortical neurons, but not in ASIC1a/2/3 triple knockout neurons. The inhibition in the wild-type was partially relieved by a drop in extracellular pH to 6. To test the consequences of ASIC-BK interaction for neuronal excitability, we compared action potential firing in cultured cortical neurons from wild-type and ASIC1a/2/3 null mice. We found that in the knockout, action potentials were narrow and exhibited increased after-hyperpolarization. Moreover, the excitability of these neurons was significantly increased. These findings are consistent with increased BK channel activity in the neurons from ASIC1a/2/3 null mice. Our data suggest that ASICs can act as endogenous pH-dependent inhibitors of BK channels, and thereby can reduce neuronal excitability.     

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed^1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere^1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms^3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous^5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects^5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms^5,8-13.Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals^14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection^13,16,17 or stable transduction^5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells²⁰. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.     

Photoperiod Modulates Fast Delayed Rectifier Potassium Currents in the Mammalian Circadian Clock

One feature of the mammalian circadian clock, situated in the suprachiasmatic nucleus (SCN), is its ability to measure day length and thereby contribute to the seasonal adaptation of physiology and behavior. The timing signal from the SCN, namely the 24 hr pattern of electrical activity, is adjusted according to the photoperiod being broader in long days and narrower in short days. Vasoactive intestinal peptide and gamma-aminobutyric acid play a crucial role in intercellular communication within the SCN and contribute to the seasonal changes in phase distribution. However, little is known about the underlying ionic mechanisms of synchronization. The present study was aimed to identify cellular mechanisms involved in seasonal encoding by the SCN. Mice were adapted to long-day (light–dark 16:8) and short-day (light–dark 8:16) photoperiods and membrane properties as well as K⁺ currents activity of SCN neurons were measured using patch-clamp recordings in acute slices. Remarkably, we found evidence for a photoperiodic effect on the fast delayed rectifier K⁺ current, that is, the circadian modulation of this ion channel’s activation reversed in long days resulting in 50% higher peak values during the night compared with the unaltered day values. Consistent with fast delayed rectifier enhancement, duration of action potentials during the night was shortened and afterhyperpolarization potentials increased in amplitude and duration. The slow delayed rectifier, transient K⁺ currents, and membrane excitability were not affected by photoperiod. We conclude that photoperiod can change intrinsic ion channel properties of the SCN neurons, which may influence cellular communication and contribute to photoperiodic phase adjustment.     

Circadian Oscillations of Molecular Clock Components in the Cerebellar Cortex of the Rat

The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum. (Author correspondence: mrath@sund.ku.dk)     

Establishment of cell lines derived from the rat suprachiasmatic nucleus

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.