Phase differences between SCN neurons and their role in photoperiodic encoding; a simulation of ensemble patterns using recorded single unit electrical activity patterns

In mammals, a major circadian pacemaker is located in the suprachiasmatic nuclei (SCN), at the base of the anterior hypothalamus. The pacemaker controls daily rhythms in behavioral, physiological and endocrine functions and is synchronized to the external light-dark cycle via the retinohypothalamic tract. The SCN are also involved in photoperiodic processes. Changes in day-length are perceived by the SCN, and result in a compression or decompression of the SCN ensemble pattern, which appears to be effectuated by changes in phase relationship among oscillating neurons. By simulation experiments, we have previously shown that the duration of the single unit activity pattern is of minor importance for the broadness of the population activity peak. Instead, the phase distribution among neurons is leading to substantial differences in the broadness of the population pattern. We now show that the combination of (i) changes in the single unit activity pattern and (ii) changes in the phase distribution among oscillating neurons is also effective to encode photoperiodic information. Moreover, we simulated the ensemble waveform of the SCN with recently recorded single unit electrical activity patterns of mice under long and short photoperiods. We show that these single unit activity patterns cannot account for changes in the population waveform of the SCN unless their phase distribution is changed. A narrow distribution encodes for short photoperiods, while a wider distribution is required to encode long photoperiods. The present studies show that recorded patterns in single unit activity rhythms, measured under long and short day conditions, can be used in simulation experiments and are informative in showing which attributes of the neuronal discharge patterns leads to the capacity of the SCN to encode photoperiod.     

Rhythmic multiunit neural activity in slices of hamster suprachiasmatic nucleus reflect prior photoperiod

The suprachiasmatic nucleus (SCN) is an endogenous circadian pacemaker, and SCN neurons exhibit circadian rhythms of electrophysiological activity in vitro. In vivo, the functional state of the pacemaker depends on changes in day length (photoperiod), but it is not known if this property persists in SCN tissue isolated in vitro. To address this issue, we prepared brain slices from hamsters previously entrained to light-dark (LD) cycles of different photoperiods and analyzed rhythms of SCN multiunit neuronal activity using single electrodes. Rhythms in SCN slices from hamsters entrained to 8:16-, 12:12-, and 14:10-h LD cycles were characterized by peak discharge rates relatively higher during subjective day than subjective night. The mean duration of high neuronal activity was photoperiod dependent, compressed in slices from the short (8:16 and 12:12 LD) photoperiods, and decompressed (approximately doubled) in slices from the long (14:10 LD) photoperiod. In slices from all photoperiods, the mean phase of onset of high neuronal activity appeared to be anchored to subjective dawn. Our results show that the electrophysiological activity of the SCN pacemaker depends on day length, extending previous in vivo data, and demonstrate that this capacity is sustained in vitro.     

Seasonal encoding by the circadian pacemaker of the SCN

The circadian pacemaker of the suprachiasmatic nucleus (SCN) functions as a seasonal clock through its ability to encode day length [1-6]. To investigate the mechanism by which SCN neurons code for day length, we housed mice under long (LD 16:8) and short (LD 8:16) photoperiods. Electrophysiological recordings of multiunit activity (MUA) in the SCN of freely moving mice revealed broad activity profiles in long days and compressed activity profiles in short days. The patterns remained consistent after release of the mice in constant darkness. Recordings of MUA in acutely prepared hypothalamic slices showed similar differences between the SCN electrical activity patterns in vitro in long and short days. In vitro recordings of neuronal subpopulations revealed that the width of the MUA activity profiles was determined by the distribution of phases of contributing units within the SCN. The subpopulation patterns displayed a significantly broader distribution in long days than in short days. Long-term recordings of single-unit activity revealed short durations of elevated activity in both short and long days (3.48 and 3.85 hr, respectively). The data indicate that coding for day length involves plasticity within SCN neuronal networks in which the phase distribution of oscillating neurons carries information on the photoperiod's duration.     

Modeling the Seasonal Adaptation of Circadian Clocks by Changes in the Network Structure of the Suprachiasmatic Nucleus

The dynamics of circadian rhythms needs to be adapted to day length changes between summer and winter. It has been observed experimentally, however, that the dynamics of individual neurons of the suprachiasmatic nucleus (SCN) does not change as the seasons change. Rather, the seasonal adaptation of the circadian clock is hypothesized to be a consequence of changes in the intercellular dynamics, which leads to a phase distribution of electrical activity of SCN neurons that is narrower in winter and broader during summer. Yet to understand this complex intercellular dynamics, a more thorough understanding of the impact of the network structure formed by the SCN neurons is needed. To that effect, we propose a mathematical model for the dynamics of the SCN neuronal architecture in which the structure of the network plays a pivotal role. Using our model we show that the fraction of long-range cell-to-cell connections and the seasonal changes in the daily rhythms may be tightly related. In particular, simulations of the proposed mathematical model indicate that the fraction of long-range connections between the cells adjusts the phase distribution and consequently the length of the behavioral activity as follows: dense long-range connections during winter lead to a narrow activity phase, while rare long-range connections during summer lead to a broad activity phase. Our model is also able to account for the experimental observations indicating a larger light-induced phase-shift of the circadian clock during winter, which we show to be a consequence of higher synchronization between neurons. Our model thus provides evidence that the variations in the seasonal dynamics of circadian clocks can in part also be understood and regulated by the plasticity of the SCN network structure.     

Phase resetting of the mammalian circadian clock relies on a rapid shift of a small population of pacemaker neurons

The circadian pacemaker of the suprachiasmatic nuclei (SCN) contains a major pacemaker for 24 h rhythms that is synchronized to the external light-dark cycle. In response to a shift in the external cycle, neurons of the SCN resynchronize with different pace. We performed electrical activity recordings of the SCN of rats in vitro following a 6 hour delay of the light-dark cycle and observed a bimodal electrical activity pattern with a shifted and an unshifted component. The shifted component was relatively narrow as compared to the unshifted component (2.2 h and 5.7 h, respectively). Curve fitting and simulations predicted that less than 30% of the neurons contribute to the shifted component and that their phase distribution is small. This prediction was confirmed by electrophysiological recordings of neuronal subpopulations. Only 25% of the neurons exhibited an immediate shift in the phase of the electrical activity rhythms, and the phases of the shifted subpopulations appeared significantly more synchronized as compared to the phases of the unshifted subpopulations (p<0.05). We also performed electrical activity recordings of the SCN following a 9 hour advance of the light-dark cycle. The phase advances induced a large desynchrony among the neurons, but consistent with the delays, only 19% of the neurons peaked at the mid of the new light phase. The data suggest that resetting of the central circadian pacemaker to both delays and advances is brought about by an initial shift of a relatively small group of neurons that becomes highly synchronized following a shift in the external cycle. The high degree of synchronization of the shifted neurons may add to the ability of this group to reset the pacemaker. The large desynchronization observed following advances may contribute to the relative difficulty of the circadian system to respond to advanced light cycles.     

Defined cell groups in the rat suprachiasmatic nucleus have different day/night rhythms of single-unit activity in vivo

The electrical activity of the rat suprachiasmatic nucleus (SCN) was examined in anesthetized rats in vivo using single-unit electrophysiological techniques. The present data confirm the daily variation in the electrical activity of the SCN previously reported in vitro and in vivo using multiple-unit recording techniques. They further suggest that subpopulations of suprachiasmatic neurons with different neural connections have a different daily rhythm of activity. Neurons in the SCN region showed a significant rhythm of activity (p = 0.034; Kruskall-Wallis analysis of variance [KW-ANOVA]). The greatest activity occurred during the second part of the light period (ZT 10-12), and the lowest activity occurred in the early part of the light period (ZT 0-2). The subgroup of cells in the suprachiasmatic region with output projections to the arcuate nucleus (ARC) and/or supraoptic nucleus (SON) regions also showed a significant rhythm (p = 0.001; K-W ANOVA). Their activity appeared to show two peaks near the light-dark (ZT 10-12) and dark-light (ZT 22-24) transition periods with the lowest activity at ZT 16-18. This rhythm was significantly different (p = 0.016) from that of neurons without an output projection to the ARC and/or SON. Retinorecipient suprachiasmatic neurons appeared to have a less robust daily rhythm in their activity. The change in the firing behavior of the cells was not reflected simply by changes in mean firing rate. Examination of the coefficient of variation of the interspike interval distribution of cells at different times of day revealed changes in the firing pattern of cells in the SCN region that did not have output projections (p = 0.032; K-W ANOVA). The present results thus suggest that the SCN is composed of a heterogeneous population of neurons and that different rhythms of activity are expressed by neurons with different neural connections. There were changes in both firing pattern and firing rate.     

SCN outputs and the hypothalamic balance of life

The circadian clock in the suprachiasmatic nucleus (SCN) is composed of thousands of oscillator neurons, each dependent on the cell-autonomous action of a defined set of circadian clock genes. Still, the major question remains how these individual oscillators are organized into a biological clock producing a coherent output able to time all the different daily changes in behavior and physiology. In the present review, the authors discuss the anatomical connections and neurotransmitters used by the SCN to control the daily rhythms in hormone release. The efferent SCN projections mainly target neurons in the medial hypothalamus surrounding the SCN. The activity of these preautonomic and neuroendocrine target neurons is controlled by differentially timed waves of, among others, vasopressin, GABA, and glutamate release from SCN terminals. Together, the data on the SCN control of neuroendocrine rhythms provide clear evidence not only that the SCN consists of phenotypically (i.e., according to neurotransmitter content) different subpopulations of neurons but also that subpopulations should be distinguished (within phenotypically similar groups of neurons) based on the acrophase of their (electrical) activity. Moreover, the specialization of the SCN may go as far as a single body structure, that is, the SCN seems to contain neurons that specifically target the liver, pineal, and adrenal.     

Circadian oscillation of the multiple unit activity in the guinea pig suprachiasmatic nucleus

Summary In the guinea pig with chronically implanted electrodes, neuronal multiple unit activity (MUA) was recorded inside and outside the suprachiasmatic nucleus (SCN). Long-term recording of the SCN indicated distinct daily rhythms with a daytime peak in MUA during a 24-h light-dark (LD 1212) cycle. On the other hand, MUA recorded from adjacent hypothalamic regions outside the SCN showed a phase reversal with a nighttime peak, similarly to the rat. The amplitude of the rhythms recorded outside the SCN was much smaller (one-half to one-quarter) than that inside the SCN. These rhythms persisted during constant darkness indicating characteristics of endogenous circadian rhythmicity. When the external lightdark cycle was delayed abruptly for 12 h, MUA rhythms showed a gradual phase shift taking 7–10 days for complete reentrainment. Overt behavior including sleep-wakefulness did not show significant and consistent daily or circadian rhythms in spite of the distinct oscillation in neuronal activity inside the SCN.Abbreviations SCN suprachiasmatic nucleus - MUA multiple unit activity     

Dissociation between circadian Per1 and neuronal and behavioral rhythms following a shifted environmental cycle

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a major circadian pacemaker that imposes or entrains rhythmicity on other structures by generating a circadian pattern in electrical activity. The identification of "clock genes" within the SCN and the ability to dynamically measure their rhythmicity by using transgenic animals open up new opportunities to study the relationship between molecular rhythmicity and other well-documented rhythms within the SCN. We investigated SCN circadian rhythms in Per1-luc bioluminescence, electrical activity in vitro and in vivo, as well as the behavioral activity of rats exposed to a 6-hr advance in the light-dark cycle followed by constant darkness. The data indicate large and persisting phase advances in Per1-luc bioluminescence rhythmicity, transient phase advances in SCN electrical activity in vitro, and an absence of phase advances in SCN behavioral or electrical activity measured in vivo. Surprisingly, the in vitro phase-advanced electrical rhythm returns to the phase measured in vivo when the SCN remains in situ. Our study indicates that hierarchical levels of organization within the circadian timing system influence SCN output and suggests a strong and unforeseen role of extra-SCN areas in regulating pacemaker function.     

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.     

Circadian Activity Rhythms and Phase‐Shifting of Cultured Neurons of the Rat Suprachiasmatic Nucleus

The mammalian suprachiasmatic nucleus (SCN) is the major endogenous pacemaker that coordinates various daily rhythms including locomotor activity and autonomous and endocrine responses, through a neuronal and humoral influence. In the present study we examined the behavior of dispersed individual SCN neurons obtained from 1‐ to 3‐day‐old rats cultured on multi‐microelectrode arrays (MEAs). SCN neurons were identified by immunolabeling for the neuropeptides arginine‐vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). Single SCN neurons cultured at low density onto an MEA can express firing rate patterns with different circadian phases. In these cultures we observed rarely synchronized firing patterns on adjacent electrodes. This suggests that, in cultures of low cell densities, SCN neurons function as independent pacemakers. To investigate whether individual pacemakers can be influenced independently by phase‐shifting stimuli, we applied melatonin (10 pM to 100 nM) for 30 min at different circadian phases and continuously monitored the firing rate rhythms. Melatonin could elicit phase‐shifting responses in individual clock cells which had no measurable input from other neurons. In several neurons, phase‐shifts occurred with a long delay in the second or third cycle after melatonin treatment, but not in the first cycle. Phase‐shifts of isolated SCN neurons were also observed at times when the SCN showed no sensitivity to these phase‐shifting stimuli in recordings from brain slices. This finding suggests that the neuronal network plays an essential role in the control of phase‐shifts.     

Signaling in the mammalian circadian clock: the NO/cGMP pathway

Mammalian circadian rhythms are generated by a hypothalamic suprachiasmatic nuclei (SCN) clock. Light pulses synchronize body rhythms by inducing phase delays during the early night and phase advances during the late night. Phosphorylation events are known to be involved in circadian phase shifting, both for delays and advances. Pharmacological inhibition of the cGMP-dependent kinase (cGK) or Ca2+/calmodulin-dependent kinase (CaMK), or of neuronal nitric oxide synthase (nNOS) blocks the circadian responses to light in vivo. Light pulses administered during the subjective night, but not during the day, induce rapid phosphorylation of both p-CAMKII and p-nNOS (specifically phosphorylated by CaMKII). CaMKII inhibitors block light-induced nNOS activity and phosphorylation, suggesting a direct pathway between both enzymes. Furthermore, SCN cGMP exhibits diurnal and circadian rhythms with maximal values during the day or subjective day. This variation of cGMP levels appears to be related to temporal changes in phosphodiesterase (PDE) activity and not to guanylyl cyclase (GC) activity. Light pulses increase SCN cGMP levels at circadian time (CT) 18 (when light causes phase advances of rhythms) but not at CT 14 (the time for light-induced phase delays). cGK II is expressed in the hamster SCN and also exhibits circadian changes in its levels, peaking during the day. Light pulses increase cGK activity at CT 18 but not at CT 14. In addition, cGK and GC inhibition by KT-5823 and ODQ significantly attenuated light-induced phase shifts at CT 18. This inhibition did not change c-Fos expression SCN but affected the expression of the clock gene per in the SCN. These results suggest a signal transduction pathway responsible for light-induced phase advances of the circadian clock which could be summarized as follows: Glu-Ca2+-CaMKII-nNOS-GC-cGMP-cGK-->-->clock genes. This pathway offers a signaling window that allows peering into the circadian clock machinery in order to decipher its temporal cogs and wheels.     

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.     

Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression

Circadian (ca. 24 hr) oscillations in expression of mammalian "clock genes" are found not only in the suprachiasmatic nucleus (SCN), the central circadian pacemaker, but also in peripheral tissues. Under constant conditions in vitro, however, rhythms of peripheral tissue explants or immortalized cells damp partially or completely. It is unknown whether this reflects an inability of peripheral cells to sustain rhythms, as SCN neurons can, or a loss of synchrony among cells. Using bioluminescence imaging of Rat-1 fibroblasts transfected with a Bmal1::luc plasmid and primary fibroblasts dissociated from mPer2(Luciferase-SV40) knockin mice, we monitored single-cell circadian rhythms of clock gene expression for 1-2 weeks. We found that single fibroblasts can oscillate robustly and independently with undiminished amplitude and diverse circadian periods. Cells were partially synchronized by medium changes at the start of an experiment, but due to different intrinsic periods, their phases became randomly distributed after several days. Closely spaced cells in the same culture did not have similar phases, implying a lack of functional coupling among cells. Thus, like SCN neurons, single fibroblasts can function as independent circadian oscillators; however, lack of oscillator coupling in dissociated cell cultures leads to a loss of synchrony among individual cells and damping of the ensemble rhythm at the population level.     

Serotonin agonist quipazine induces photic-like phase shifts of the circadian activity rhythm and c-Fos expression in the rat suprachiasmatic nucleus

Nonphotic stimuli can reset and entrain circadian activity rhythms in hamsters and mice, and serotonin is thought to be involved in the phase-resetting effects of these stimuli. In the present study, the authors examined the effect of the serotonin agonist quipazine on circadian activity rhythms in three inbred strains of rats (ACI, BH, and LEW). Furthermore, they investigated the effect of quipazine on the expression of c-Fos in the mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN). Quipazine reduced the amount of running wheel activity for 3 h after treatment, however, no long-term changes in tau and in the activity level were observed. More important, quipazine induced significant phase advances of the activity rhythm and c-Fos production in the SCN at the end of the subjective night (Circadian Time [CT] 22), whereas neither phase shifts nor c-Fos induction were observed during the subjective day. Quipazine injections also resulted in moderate phase delays at the beginning of the subjective night (CT 14). A similar phase-response characteristic typically can be observed for photic stimuli. By contrast, nonphotic stimuli normally produce phase advances during the subjective day. The present results suggest species differences between the hamster and the rat with respect to the serotonergic action on circadian timekeeping and indicate that serotonergic pathways play a role in the transmission of photic information to the SCN of rats.     

Bimodal clock gene expression in mouse suprachiasmatic nucleus and peripheral tissues under a 7-hour light and 5-hour dark schedule

Using the mPer1::luc real-time monitoring technique, the authors observed the bimodal patterns of mPer1 bioluminescence on each side of the SCN, in parallel with maintaining synchronization between the left and right sides of the SCN under an artificial light:dark:light:dark (LDLD) 7:5:7:5 condition. In situ hybridization analysis of mPer1 and mBmal1 mRNA distribution in the SCN showed that in 1 photophase (morning photophase; M) of LDLD, the mPer1 level in the ventrolateral-like (VL-like) subdivision of the SCN was higher than that in the dorsomedial-like (DM-like) subdivision, and this regional distribution pattern was reversed in another photophase (evening photophase; E). In contrast, the mBmal1 level was higher in the DM-like subdivision than in the VL-like subdivision in the M phase, and this distribution changed in the E phase. The prokineticin 2 (PK2) mRNA that encodes an SCN output molecule that is thought to transmit the circadian locomotor rhythms was reduced in both the DM-like and VL-like SCN and did not clearly correlate with the activity under the LDLD condition. The expression of mPer1 and mPer2 in the liver was clearly bimodal, whereas the expressions of other clock genes were not synchronized to the LDLD condition. These results may provide important insights into the mechanism underlying the splitting or bimodal rhythms that may in turn facilitate the understanding of the ability to measure the seasonal day length in mammals.     

Circadian dynamics of cytosolic and nuclear Ca2+ in single suprachiasmatic nucleus neurons

Intracellular free Ca(2+) regulates diverse cellular processes, including membrane potential, neurotransmitter release, and gene expression. To examine the cellular mechanisms underlying the generation of circadian rhythms, nucleus-targeted and untargeted cDNAs encoding a Ca(2+)-sensitive fluorescent protein (cameleon) were transfected into organotypic cultures of mouse suprachiasmatic nucleus (SCN), the primary circadian pacemaker. Circadian rhythms in cytosolic but not nuclear Ca(2+) concentration were observed in SCN neurons. The cytosolic Ca(2+) rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode array, with a mean advance in phase of 4 hr. Tetrodotoxin blocked MUA, but not Ca(2+) rhythms, while ryanodine damped both Ca(2+) and MUA rhythms. These results demonstrate cytosolic Ca(2+) rhythms regulated by the release of Ca(2+) from ryanodine-sensitive stores in SCN neurons.     

Cellular communication and coupling within the suprachiasmatic nucleus

In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a pair of structures in the hypothalamus known as the suprachiasmatic nucleus (SCN). Importantly, when SCN neurons are removed from the organism and maintained in a brain slice preparation, they continue to generate 24h rhythms in electrical activity, secretion, and gene expression. Previous studies suggest that the basic mechanism responsible for the generation of these rhythms is intrinsic to individual cells in the SCN. If we assume that individual cells in the SCN are competent circadian oscillators, it is obviously important to understand how these cells communicate and remain synchronized with each other. Cell-to-cell communication is clearly necessary for conveying inputs to and outputs from the SCN and may be involved in ensuring the high precision of the observed rhythm. In addition, there is a growing body of evidence that a number of systems-level phenomena could be dependent on the cellular communication between circadian pacemaker neurons. It is not yet known how this cellular synchronization occurs, but it is likely that more than one of the already proposed mechanisms is utilized. The purpose of this review is to summarize briefly the possible mechanisms by which the oscillatory cells in the SCN communicate with each other. (Chronobiology International, 18(4)579-600, 2001)