In search of the pathways for light-induced pacemaker resetting in the suprachiasmatic nucleus

Within the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is a circadian pacemaker that functions as a clock. Its endogenous period is adjusted to the external 24-h light-dark cycle, primarily by light-induced phase shifts that reset the pacemaker's oscillation. Evidence using a wide variety of neurobiological and molecular genetic tools has elucidated key elements that comprise the visual input pathway for SCN photoentrainment in rodents. Important questions remain regarding the intracellular signals that reset the autoregulatory molecular loop within photoresponsive cells in the SCN's retino-recipient subdivision, as well as the intercellular coupling mechanisms that enable SCN tissue to generate phase shifts of overt behavioral and physiological circadian rhythms such as locomotion and SCN neuronal firing rate. Multiple neurotransmitters, protein kinases, and photoinducible genes add to system complexity, and we still do not fully understand how dawn and dusk light pulses ultimately produce bidirectional, advancing and delaying phase shifts for pacemaker entrainment.     

Light-induced c-Fos expression in the SCN and behavioural phase shifts of Djungarian hamsters with a delayed activity onset

C-Fos expression in the suprachiasmatic nucleus (SCN) and phase shifts of the activity rhythm following photic stimulation were investigated in Djungarian hamsters (Phodopus sungorus) of two different circadian phenotypes. Wild-type (WT) hamsters display robust daily patterns of locomotor activity according to the light/dark conditions. Hamsters of the DAO (delayed activity onset) phenotype, however, progressively delay the activity onset, whereas activity offset remains coupled to “light-on”. Although the exact reason for the delayed activity onset is not yet clarified, it is connected with a disturbed interaction between the light/dark cycle and the circadian clock. The aim was to test the link between photoreception and the behavioral output of the circadian system in hamsters of both phenotypes, to get further insight in the underlying mechanism of the DAO phenomenon. Animals were exposed to short light pulses at different times during the dark period to analyze phase shifts of the activity rhythm and expression of Fos protein in the SCN. The results indicate that the photosensitive phase in DAO hamsters is shifted like the activity onset. Also, phase shifts were significantly smaller in DAO hamsters. At the same time, levels of Fos expression did not differ between phenotypes regarding the circadian phase. The results provide evidence that the shifted photosensitivity of the circadian system in DAO hamsters does not differ from that of WT animals, and lead us to conclude that processes within the SCN that enable light information to reset the circadian pacemaker might offer an explanation for the DAO phenomenon.     

Circadian rhythms in Neurospora: a new measurement, the reset zone

The authors define a new feature of a circadian rhythm, the reset zone, and point out its usefulness for predictions concerning oscillator behavior. The reset zone measures the responses of a circadian system to resetting pulses. It can be easily determined from a phase transition curve (PTC), which is simply a phase response curve (PRC) replotted as new phase versus old phase (Winfree's format). The reset zone is the range of new phases seen in such a plot and has two potentially useful characteristics: its size and its midpoint. A series of experiments with Neurospora involving temperature pulses indicated that the size of the reset zone changed in a nonlinear way in response to both the duration of 40 degrees C pulses and to the magnitude of temperature change for 3-h pulses. Other existing data are replotted to show how the reset zone size varies with growth temperature and with the period of different clock mutants. Employing exclusively reset zone data within the framework of a limit cycle displacement model, an equation is formulated that predicts the relative changes in the values of state variables of the oscillator for changes in any given environmental condition, such as temperature. Examples are also drawn from other organisms, such as hamsters, Gonyalaux, Kalanchoe, and Drosophila, illustrating the usefulness of the reset zone measurement. It can be used as a numerical scale for assessing the strength of a pulse, for comparing the relative effects of a given pulse applied to different organisms or mutants, for determining the directionality of the changes in state variables produced by various types of pulses, and possibly for measuring clock amplitude.     

Photic resetting of the human circadian pacemaker in the absence of conscious vision

Ocular light exposure patterns are the primary stimuli for entraining the human circadian system to the local 24-h day. Many totally blind persons cannot use these stimuli and, therefore, have circadian rhythms that are not entrained. However, a few otherwise totally blind persons retain the ability to suppress plasma melatonin concentrations after ocular light exposure, probably using a neural pathway that includes the site of the human circadian pacemaker, suggesting that light information is reaching this site. To test definitively whether ocular light exposure could affect the circadian pacemaker of some blind persons and whether melatonin suppression in response to bright light correlates with light-induced phase shifts of thecircadian system, the authorsperformed experiments with 5 totally blind volunteers using a protocol known to induce phase shifts of the circadian pacemaker in sighted individuals. In the 2 blind individuals who maintained light-induced melatonin suppression, the circadian system was shifted by appropriately timed bright-light stimuli. These data demonstrate that light can affect the circadian pacemaker of some totally blind individuals--either by altering the phase of the circadian pacemaker or by affecting its amplitude. They are consistent with data from animal studies demonstrating that there are different neural pathways and retinal cells that relay photic information to the brain: one for conscious light perception and the other for non-image-forming functions.     

The effect of repeated pulses of light at the same time on period responses of the rat circadian pacemaker

A consequence of simple velocity-based models is that, in response to light pulses, the circadian period should adjust inversely to phase. In addition, because of the interaction of circadian period and phase response, earlier circadian period changes should modify later circadian period changes. The literature contains few mentions of response curves of circadian period responses following light pulses. Rats were exposed to four pulses of light (60 minutes, 1000 lux) at the same circadian time, a minimum of 26 days apart; we assessed period responses and possible bias in the period-response curve. Modulation of circadian period following light-induced phase responses was examined by assessing the period of running wheel activity onset. Phase and circadian period were not consistently found to share an inverse relationship. Moreover, biases in initial period tended to be increased by the experimental protocol regardless of circadian time of pulse. Rats with a short initial (high-velocity) period had a lengthened period, while rats with a long initial period (low velocity) tended to have a reduce period. However, rats with a long initial period were phase delay biased, not phase advance biased. These results do not support a simple velocity model of the pacemaker. (Chronobiology International, 18(2), 187-201, 2001)     

Asymmetric re‐entrainment and phase response of the circadian activity rhythm of a nocturnal marsupial (Petaurus breviceps: Phalangeridae)

Abstract

Sugar Gliders (Petaurus breviceps) re‐entrain faster after 8‐h delay shifts of an LD 12:12 and an LD 8:16 (31–56:0.3 lux each) than after 8‐h advance shifts of these Zeitgeber cycles. In order to test whether this asymmetric re‐entrainment behavior is related to, or even caused by the phase response characteristics of the circadian system, the phase response of the activity rhythm to short and long light pulses was studied. Short light pulses (15 min of 31–56 lux against a background intensity of 0.3 lux) caused only relatively small delay shifts when applied around the onset, and more pronounced advance shifts when given at the end of the activity time (α). Onset and end of activity shifted by different amounts. Long light pulses produced by 8‐h advances and delays of one single lighttime of an LD 12:12 elicited pronounced phase delays when applied at the beginning of the activity time, but only minor phase advances when given at the posterior part of α. These results indicate that in Petaurus breviceps the phase response characteristics to long light pulses exerting parametric effects of light are responsible for the pronounced asymmetry effect in re‐entrainment. Differing phase responses of onset and end of activity point to a two‐oscillator structure of the circadian pacemaker system in this marsupial.     

Novel phase-shifting effects of GABAA receptor activation in the suprachiasmatic nucleus of a diurnal rodent

The vast majority of neurons in the suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals, contain the inhibitory neurotransmitter GABA. Most studies investigating the role of GABA in the SCN have been performed using nocturnal rodents. Activation of GABA(A) receptors by microinjection of muscimol into the SCN phase advances the circadian activity rhythm of nocturnal rodents, but only during the subjective day. Nonphotic stimuli that reset the circadian pacemaker of nocturnal rodents also produce phase advances during the subjective day. The role of GABA in the SCN of diurnal animals and how it may differ from nocturnal animals is not known. In the studies described here, the GABA(A) agonist muscimol was microinjected directly into the SCN region of diurnal unstriped Nile grass rats (Arvicanthis niloticus) at various times in their circadian cycle. The results demonstrate that GABA(A) receptor activation produces large phase delays during the subjective day in grass rats. Treatment with TTX did not affect the ability of muscimol to induce phase delays, suggesting that muscimol acts directly on pacemaker cells within the SCN. These data suggest that the circadian pacemakers of nocturnal and diurnal animals respond to the most abundant neurochemical signal found in SCN neurons in opposite ways. These findings are the first to demonstrate a fundamental difference in the functioning of circadian pacemaker cells in diurnal and nocturnal animals.     

Phase shifting the hamster circadian clock by 15-minute dark pulses

The mammalian circadian pacemaker can be phase shifted by exposure to a period of darkness interrupting otherwise continuous light. Circadian phase shifting by dark pulses was interpreted originally as reflecting a photic mirror-image mechanism, but more recent observations suggest that dark pulse-induced phase shifting may be mediated by a nonphotic, behavioral state-dependent mechanism. The authors recently presented evidence indicating that the dark-pulse phase response curve (PRC) is in fact a complex function, reflecting both photic mirror image and nonphotic mechanisms at different phases of the circadian cycle. Previous studies of dark pulse-induced phase shifting have universally employed relatively long (2 to 6 h) pulses, which complicates PRC analysis due to the extended segment of the underlying PRC spanned by such a long pulse. The present study was therefore designed to examine the phase-shifting effects of brief 15-min dark pulses presented at both mid-subjective day and subjective dusk, and to explore the possible activity dependence of these effects by using physical restraint to prevent evoked locomotor activity. The results indicate that 15-min dark pulses are effective phase-shifting stimuli at both midday and dusk. Furthermore, as with longer dark pulses, phase shifting by 15-min dark pulses is completely blocked by physical restraint during subjective day but combines in a simple additive manner with the independent phase-shifting effect of restraint at subjective dusk.     

Serotonergic Actions and Interactions on the SCN Circadian Pacemaker: In Vitro Investigations

The phase of the mammalian circadian pacemaker located in the suprachiasmatic nuclei (SCN) is controlled by a multitude of stimuli. While phase control is undoubtedly dominated by photic input, the serotonergic input from the raphe nuclei also influences SCN clock phase. In this article I review the evidence for serotonergic modulation of the SCN pacemaker, and the cellular mechanisms underlying these effects, obtained from in vitro experiments performed during the past decade. Serotonin can advance the SCN pacemaker when applied during the subjective day, and delay the pacemaker when applied during the subjective night. The daytime advances appear due to stimulation of 5HT₇ receptors, activation of adenylate cyclase and protein kinase A, and opening of K⁺ channels. The synthesis of new proteins may also be critical for these phase shifts. Serotonergic phase advances can be inhibited by a variety of other modulatory inputs to the SCN, including neuropeptide Y, melatonin, and glutamate. Together, these data demonstrate that SCN circadian pacemaker phase is controlled by a complex interplay between multiple afferent stimuli, and that serotonin plays a critical role in this process.     

Efficacy of a single sequence of intermittent bright light pulses for delaying circadian phase in humans

It has been shown in animal studies that exposure to brief pulses of bright light can phase shift the circadian pacemaker and that the resetting action of light is most efficient during the first minutes of light exposure. In humans, multiple consecutive days of exposure to brief bright light pulses have been shown to phase shift the circadian pacemaker. The aim of the present study was to determine whether a single sequence of brief bright light pulses administered during the early biological night would phase delay the human circadian pacemaker. Twenty-one healthy young subjects underwent a 6.5-h light exposure session in one of three randomly assigned conditions: 1) continuous bright light of approximately 9,500 lux, 2) intermittent bright light (six 15-min bright light pulses of approximately 9,500 lux separated by 60 min of very dim light of <1 lux), and 3) continuous very dim light of <1 lux. Twenty subjects were included in the analysis. Core body temperature (CBT) and melatonin were used as phase markers of the circadian pacemaker. Phase delays of CBT and melatonin rhythms in response to intermittent bright light pulses were comparable to those measured after continuous bright light exposure, even though the total exposure to the intermittent bright light represented only 23% of the 6.5-h continuous exposure. These results demonstrate that a single sequence of intermittent bright light pulses can phase delay the human circadian pacemaker and show that intermittent pulses have a greater resetting efficacy on a per minute basis than does continuous exposure.     

Light-Induced resetting of the circadian pacemaker: quantitative analysis of transient versus steady-state phase shifts.

The suprachiasmatic nuclei of the hypothalamus contain the major circadian pacemaker in mammals, driving circadian rhythms in behavioral and physiological functions. This circadian pacemaker's responsiveness to light allows synchronization to the light-dark cycle. Phase shifting by light often involves several transient cycles in which the behavioral activity rhythm gradually shifts to its steady-state position. In this article, the authors investigate in Syrian hamsters whether a phase-advancing light pulse results in immediate shifts of the PRC at the next circadian cycle. In a first series of experiments, the authors aimed a light pulse at CT 19 to induce a phase advance. It appeared that the steady-state phase advances were highly correlated with activity onset in the first and second transient cycle. This enabled them to make a reliable estimate of the steady-state phase shift induced by a phase-advancing light pulse on the basis of activity onset in the first transient cycle. In the next series of experiments, they presented a light pulse at CT 19, which was followed by a second light pulse aimed at the delay zone of the PRC on the next circadian cycle. The immediate and steady-state phase delays induced by the second light pulse were compared with data from a third experiment in which animals received a phase-delaying light pulse only. The authors observed that the waveform of the phase-delay part of the PRC (CT 12-16) obtained in Experiment 2 was virtually identical to the phase-delay part of the PRC for a single light pulse (obtained in Experiment 3). This finding allowed for a quantitative assessment of the data. The analysis indicates that the delay part of the PRC-between CT 12 and CT 16-is rapidly reset following a light pulse at CT 19. These findings complement earlier findings in the hamster showing that after a light pulse at CT 19, the phase-advancing part of the PRC is immediately shifted. Together, the data indicate that the basis for phase advancing involves rapid resetting of both advance and delay components of the PRC.     

Circadian phase shifting induced by clonidine injections in Syrian hamsters

Abstract

The mammalian circadian pacemaker can be phase shifted by photic, pharmacological, and behaviorally‐derived stimuli. The phase‐response curves (PRCs) characterizing these diverse stimuli may comprise two distinct families; a photic PRC typified by the response to brief light pulses, and a non‐photic PRC, typified by the response to dark pulses and to behavioral activation. The present study examined the phase shifting effects of acute systemic treatment with the alpha2‐adrenoceptor agonist, clonidine, in Syrian hamsters. Clonidine injections (0.25 mg/kg, ip) delivered during subjective night mimicked the phase shifting effects of light pulses in animals housed in both constant darkness (DD) and constant red light (RR), but similar effects were not seen in saline‐treated controls. Both clonidine and saline injections resulted in phase advances during subjective day, but only in RR‐housed animals. Clonidine‐induced phase shifting was dose‐dependent, but rather high doses were required to induce phase shifts. Pretreatment with the selective noradrenergic neurotoxin, DSP‐4, blocked clonidine‐induced phase shifting. These results suggest that clonidine acts at presynaptic alpha2‐adrenergic autoreceptors to disinhibit spontaneous and/or evoked activity in the photic entrainment pathway.     

Entrainment of circadian clocks in mammals by arousal and food

Circadian rhythms in mammals are regulated by a system of endogenous circadian oscillators (clock cells) in the brain and in most peripheral organs and tissues. One group of clock cells in the hypothalamic SCN (suprachiasmatic nuclei) functions as a pacemaker for co-ordinating the timing of oscillators elsewhere in the brain and body. This master clock can be reset and entrained by daily LD (light-dark) cycles and thereby also serves to interface internal with external time, ensuring an appropriate alignment of behavioural and physiological rhythms with the solar day. Two features of the mammalian circadian system provide flexibility in circadian programming to exploit temporal regularities of social stimuli or food availability. One feature is the sensitivity of the SCN pacemaker to behavioural arousal stimulated during the usual sleep period, which can reset its phase and modulate its response to LD stimuli. Neural pathways from the brainstem and thalamus mediate these effects by releasing neurochemicals that inhibit retinal inputs to the SCN clock or that alter clock-gene expression in SCN clock cells. A second feature is the sensitivity of circadian oscillators outside of the SCN to stimuli associated with food intake, which enables animals to uncouple rhythms of behaviour and physiology from LD cycles and align these with predictable daily mealtimes. The location of oscillators necessary for food-entrained behavioural rhythms is not yet certain. Persistence of these rhythms in mice with clock-gene mutations that disable the SCN pacemaker suggests diversity in the molecular basis of light- and food-entrainable clocks.     

Acute light exposure suppresses circadian rhythms in clock gene expression

Light can induce arrhythmia in circadian systems by several weeks of constant light or by a brief light stimulus given at the transition point of the phase response curve. In the present study, a novel light treatment consisting of phase advance and phase delay photic stimuli given on 2 successive nights was used to induce circadian arrhythmia in the Siberian hamster ( Phodopus sungorus). We therefore investigated whether loss of rhythms in behavior was due to arrhythmia within the suprachiasmatic nucleus (SCN). SCN tissue samples were obtained at 6 time points across 24 h in constant darkness from entrained and arrhythmic hamsters, and per1, per2 , bmal1, and cry1 mRNA were measured by quantitative RT-PCR. The light treatment eliminated circadian expression of clock genes within the SCN, and the overall expression of these genes was reduced by 18% to 40% of entrained values. Arrhythmia in per1, per2, and bmal1 was due to reductions in the amplitudes of their oscillations. We suggest that these data are compatible with an amplitude suppression model in which light induces singularity in the molecular circadian pacemaker.     

Spontaneous Activity in Crustacean Neurons

Single units which discharged with regular spontaneous rhythms without intentional stimulation were observed in the ventral nerve cord by intracellular recording close to the sixth abdominal ganglion. These units were divided into two groups: group A units in which interspike intervals varied less than 10 msec.; group B units in which interspike intervals varied within a range of 10 to 30 msec. Group A units maintained "constant" interspike intervals and could not be discharged by sensory inputs, while the majority of group B units could be discharged by appropriate sensory nerve stimulation. Both group A and B units discharged to direct stimulation when the stimulating and recording electrodes were placed in the same ganglionic intersegment, and directly evoked single spikes reset the spontaneous rhythm. In group B units, presynaptic volleys reset the spontaneous rhythm of some units; but in others, synaptically evoked spikes were interpolated within the spontaneous rhythm without resetting. The phenomenon of enhancement could also be demonstrated in spontaneously active units as a result of repetitive stimulation. It is concluded that endogenous pacemaker activity is responsible for much of the regular spontaneous firing observed in crayfish central neurons, and that interaction of evoked responses with such pacemaker sites can produce a variety of effects dependent upon the anatomical relationships between pacemaker and synaptic regions.     

Serotonergic Actions and Interactions on the SCN Circadian Pacemaker: In Vitro Investigations

The phase of the mammalian circadian pacemaker located in the suprachiasmatic nuclei (SCN) is controlled by a multitude of stimuli. While phase control is undoubtedly dominated by photic input, the serotonergic input from the raphe nuclei also influences SCN clock phase. In this article I review the evidence for serotonergic modulation of the SCN pacemaker, and the cellular mechanisms underlying these effects, obtained from in vitro experiments performed during the past decade. Serotonin can advance the SCN pacemaker when applied during the subjective day, and delay the pacemaker when applied during the subjective night. The daytime advances appear due to stimulation of 5HT₇ receptors, activation of adenylate cyclase and protein kinase A, and opening of K⁺ channels. The synthesis of new proteins may also be critical for these phase shifts. Serotonergic phase advances can be inhibited by a variety of other modulatory inputs to the SCN, including neuropeptide Y, melatonin, and glutamate. Together, these data demonstrate that SCN circadian pacemaker phase is controlled by a complex interplay between multiple afferent stimuli, and that serotonin plays a critical role in this process.     

Desynchronization of brain rhythms with soft phase-resetting techniques

 Composite stimulation techniques are presented here which are based on a soft (i.e., slow and mild) reset. They effectively desynchronize a cluster of globally coupled phase oscillators in the presence of noise. A composite stimulus contains two qualitatively different stimuli. The first stimulus is either a periodic pulse train or a smooth, sinusoidal periodic stimulus with an entraining frequency close to the cluster's natural frequency. In the course of several periods of the entrainment, the cluster's dynamics is reset (restarted), independently of its initial dynamic state. The second stimulus, a single pulse, is administered with a fixed delay after the first stimulus in order to desynchronize the cluster by hitting it in a vulnerable state. The incoherent state is unstable, and thus the desynchronized cluster starts to resynchronize. Nevertheless, resynchronization can effectively be blocked by repeatedly delivering the same composite stimulus. Previously designed stimulation techniques essentially rely on a hard (i.e., abrupt) reset. With the composite stimulation techniques based on a soft reset, an effective desynchronization can be achieved even if strong, quickly resetting stimuli are not available or not tolerated. Accordingly, the soft methods are very promising for applications in biology and medicine requiring mild stimulation. In particular, it can be applied to effectively maintain incoherency in a population of oscillatory neurons which try to synchronize their firing. Accordingly, it is explained how to use the soft techniques for (i) an improved, milder, and demand-controlled deep brain stimulation for patients with Parkinson's disease or essential tremor, and for (ii) selectively blocking gamma activity in order to manipulate visual binding. Received: 3 July 2001 / Accepted in revised form: 7 February 2002     

Light pulses suppress responsiveness within the mouse photic entrainment pathway

We have characterized a decrease in photic responsiveness of the mammalian circadian entrainment pathway caused by light stimulation. Phase delays of the running-wheel activity rhythm were used to quantify the photic responsiveness of the circadian system in mice (C57BL/6J). In an initial experiment, the authors measured the responsiveness to single "saturating" light pulses ("white" fluorescent light; approximately 1876 [microW; 15 min). In two additional experiments, the authors measured responses to this stimulus at several time points following a saturating pulse at CT 14 or CT 16. Data from these experiments were analyzed in two manners. Experiment 2 was analyzed assuming that the phase of the circadian pacemaker was unchanged by an initial pulse, and Experiment 3 was analyzed assuming that the initial pulse induced an instantaneous phase delay. Results reveal a significant reduction in responsivity to light that persists for at least 2 h and possibly up to 4 h after the initial stimulus. Immediately after the stimulus, the responsiveness of the photic entrainment pathway was reduced to levels < or = 12% of normal. After 2 h, the responsiveness was < or = 42% of normal, and by 4 h, responsiveness had recovered to levels that were < or = 60% of normal (levels not statistically different from controls). By the following circadian cycle, responsiveness was more completely recovered, although the magnitude of some phase delays remained < or = 85% of normal. These major reductions in the magnitude of phase delays (and phase response curve amplitude) caused by saturating light pulses confound interpretations of two-pulse experiments designed to measure the rate of circadian phase delays. In addition, the time course for this reduced responsiveness may reflect the time course of cellular and molecular events that underlie light-induced resetting of the mammalian circadian pacemaker.     

Resetting of the hamster circadian system by dark pulses

Circadian rhythms of animals are reset by exposure to light as well as dark; however, although the parameters of photic entrainment are well characterized, the phase-shifting actions of dark pulses are poorly understood. Here, we determined the tonic and phasic effects of short (0.25 h), moderate (3 h), and long (6-9 h) duration dark pulses on the wheel-running rhythms of hamsters in constant light. Moderate- and long-duration dark pulses phase dependently reset behavioral rhythms, and the magnitude of these phase shifts increased as a function of the duration of the dark pulse. In contrast, the 0.25-h dark pulses failed to evoke consistent effects at any circadian phase tested. Interestingly, moderate- and long-dark pulses elevated locomotor activity (wheel-running) on the day of treatment. This induced wheel-running was highly correlated with phase shift magnitude when the pulse was given during the subjective day. This, together with the finding that animals pulsed during the subjective day are behaviorally active throughout the pulse, suggests that both locomotor activity and behavioral activation play an important role in the phase-resetting actions of dark pulses. We also found that the robustness of the wheel-running rhythm was weakened, and the amount of wheel-running decreased on the days after exposure to dark pulses; these effects were dependent on pulse duration. In summary, similarly to light, the resetting actions of dark pulses are dependent on both circadian phase and stimulus duration. However, dark pulses appear more complex stimuli, with both photic and nonphotic resetting properties.     

Djungarian hamsters: a species with a labile circadian pacemaker? Arrhythmicity under a light-dark cycle induced by short light pulses

In most cases, phase-shifting effects of light pulses are studied in animals kept in constant darkness (DD) or in animals released into DD following the stimulus. In this study, the authors exposed Djungarian hamsters (Phodopus sungorus) to short light pulses during the dark phase of a 16:8 light-dark (LD) cycle and thus obtained a type VI phase response curve. Light pulses early in the night caused phase delays of the activity onset as well as phase advances of the activity offset, whereas light pulses later in the night resulted in phase advances of the activity offset only. A combination of two 15-min light pulses-the first one given late in the scotophase and the second given early in the dark phase of the following night-led to a strong compression of the activity phase alpha. In 75% of all animals, daily rhythms were no longer visible after complete alpha compression, and long-term arrhythmicity (up to 145 days) persisted despite continued exposure to an LD cycle. Because three independent output rhythms of the clock (i.e., activity, body temperature, and melatonin rhythms) were equally affected, the authors conclude that overt arrhythmicity was due not merely to disrupted output pathways but to an altered state of the central pacemaker. The authors suggest a qualitative two-oscillator model to explain this phenomenon. Their hypothesis assumes that, due to loose coupling, the pacemaker of Djungarian hamsters can be driven to a state of zero phase difference between the two oscillators, with zero amplitude of their outputs.