Circadian Phase Shifting Associated with Routine Cage Maintenance: A Retrospective Analysis

Stimuli that evoke behavioral activation can phase-shift free-running circadian activity rhythms in Syrian hamsters. Activation-induced phase shifting is characterized by a phase-response curve (PRC) that is dissimilar to the PRC for photic phase shifting, and recent studies indicate that complex interactions may occur between photic and non-photic phase shifting. Since animals in the laboratory may be exposed to both photic and behaviorally activating stimulation during routine cage maintenance procedures, we performed a retrospective analysis of possible phase shifts associated with cage cleaning in individually housed hamsters maintained in either constant darkness (DD) or dim red light (RR) during the course of an ongoing study of drug-induced phase shifting. All cage cleanings were conducted under RR and were separated from drug treatments by at least one week. The results indicated that both photic and non-photic phase shifts could be induced by routine cage maintenance procedures, depending on the circadian timing of the procedure, on lighting conditions, and on the degree of evoked activity.     

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.     

Non-photic modulation of phase shifts to long light pulses

Circadian rhythms can be reset by both photic and non-photic stimuli. Recent studies have used long light exposure to produce photic phase shifts or to enhance non-photic phase shifts. The presence or absence of light can also influence the expression of locomotor rhythms through masking; light during the night attenuates locomotor activity, while darkness during the day induces locomotor activity in nocturnal animals. Given this dual role of light, the current study was designed to examine the relative contributions of photic and non-photic components present in a long light pulse paradigm. Mice entrained to a light/dark cycle were exposed to light pulses of various durations (0, 3, 6, 9, or 12 h) starting at the time of lights-off. After the light exposure, animals were placed in DD and were either left undisturbed in their home cages or had their wheels locked for the remainder of the subjective night and subsequent subjective day. Light treatments of 6, 9, and 12 h produced large phase delays. These treatments were associated with decreased activity during the nocturnal light and increased activity during the initial hours of darkness following light exposure. When the wheels were locked to prevent high-amplitude activity, the resulting phase delays to the light were significantly attenuated, suggesting that the activity following the light exposure may have contributed to the overall phase shift. In a second experiment, telemetry probes were used to assess what effect permanently locking the wheels had on the phase shift to the long light pulses. These animals had phase shifts fully as large as animals without any form of wheel lock, suggesting that while non-photic events can modulate photic phase shifts, they do not play a role in the full phase-shift response observed in animals exposed to long light pulses. This paradigm will facilitate investigations into non-photic responses of the mouse circadian system.     

Double-pulse experiments with nonphotic and photic phase-shifting stimuli.

Three-hour pulses of novelty-induced wheel running in the early to middle subjective day of golden hamsters produced phase advances of 2-3 hr. This phase shifting could be almost totally abolished by a light pulse following within 3 hr of the exercise pulse. When light pulses occurred about 8 hr after the exercise pulses, the phase-advancing effects of the latter were enhanced. Consideration of the amplitude of the phase response curve (PRC) for light pulses alone, in the test paradigms used here, showed that nonphotic and photic phase shifts did not combine additively. Antagonistic and synergistic interactions between photic and nonphotic shifts may have to be taken into account if it transpires that exercise in people can be used to assist adjustment to new schedules after crossing time zones, or in shiftwork.     

Effects of food deprivation on locomotor activity, plasma glucose, and circadian clock resetting in Syrian hamsters

Circadian rhythms in Syrian hamsters can be phase advanced by activity or arousal stimulated during the daily rest phase ("subjective day"). A widely used method for stimulating activity is confinement to a novel wheel. Some hamsters decline to run, and some procedures may reduce the probability of running. The authors evaluated food deprivation (FD) as a method to promote running. Given evidence that perturbations of cell metabolism or glucose availability may affect circadian clock function in some tissues or species, they also assessed the effects of FD on free-running circadian phase, resetting responses to photic and nonphotic stimuli and plasma glucose. In constant light, a 27-h fast significantly increased running in a novel wheel and marginally increased the average size of resulting phase shifts. FD, without novel wheel confinement, was associated with some very large phase shifts or disruption of rhythmicity in hamsters that spontaneously ran in their home wheels during the subjective day. Hamsters that ran only during the usual active phase (subjective night) or that were prevented from running did not exhibit phase shifts, despite refeeding in the mid-subjective day. Using an Aschoff Type II design for measuring shifts, a 27-h fast significantly increased the number of hamsters that ran continuously when confined to a novel wheel but did not affect the dose-response relation between the amount of running and the size of the resulting shift. A day of fasting also did not affect the size of phase delay or advance shifts to 30-min light pulses in the subjective night. Plasma glucose was markedly reduced by wheel running in combination with fasting but was increased by running in nonfasted hamsters. These results establish FD as a useful tool for stimulating activity in home cage or novel wheels and indicate that in Syrian hamsters, significant alterations in glucose availability, associated with running, fasting, and refeeding, have surprisingly little effect on circadian pacemaker function.     

A Noradrenergic Mechanism Influences the Circadian Timing System in Rats and Hamsters

Brainstem monoaminergic projections to the suprachiasmatic nucleus (SCN), and to the intergeniculate leaflet (IGL), appear to modulate both photic and non-photic effects on the circadian system. Recent work in this laboratory has concentrated on the role of noradrenaline in the regulation of circadian period and phase. Previously, this lab has shown that chronic administration of the alpha₂ adrenergic agonist, clonidine, to rats maintained in constant light (LL) shortens free-running circadian period and promotes dissociation of rhythmicity, while acute clonidine administration to hamsters produces phase shifts similar to those observed with photic stimuli. These results suggest an interaction between clonidine and photic input on circadian rhythmicity, and so the present study was designed to examine systematically the relationship between chronic clonidine administration and photic input in both rats and hamsters. In DD and low intensity LL, clonidine did not alter free-running circadian wheel-running rhythms of rats, but under moderate to high intensity LL, clonidine significantly reduced the period-lengthening effects of LL. Chronic clonidine administration also altered several aspects of circadian phase in hamsters; phase shifts in response to light pulses of varying intensity at CT 19 were reduced; steady-state entrainment phase under a 24-h light-dark cycle (LD 14:10)was delayed; and synchronization to a 23-h light-dark cycle (LD 13:10) was impaired. Clonidine appeared to have little effect on free-running period of hamsters, but a trend towards dissociation of rhythmicity under LL was observed. These effects may reflect an action of clonidine at the photic input pathways to the circadian system, or directly at the circadian pacemaker, since alpha 2 adrenoceptors have been localized both in the suprachiasmatic nucleus (SCN) and in several of its projection areas. As both clinical and experimental studies suggest that clonidine may have depressogenic properties, chronic administration of clonidine to rodents may provide an animal model of the alterations in circadian rhythmicity seen in human depression.     

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.     

Increased photic sensitivity for phase resetting but not melatonin suppression in Siberian hamsters under short photoperiods

Light regulates a variety of behavioral and physiological processes, including activity rhythms and hormone secretory patterns. Seasonal changes in the proportion of light in a day (photoperiod) further modulate those functions. Recently, short (SP) versus long days (LP) were found to markedly increase light sensitivity for phase shifting in Syrian hamsters. To our knowledge, photoperiod effects on light sensitivity have not been studied in other rodents, nor is it known if they generalize to other circadian responses. We tested whether photic phase shifting and melatonin suppression vary in Siberian hamsters maintained under LP or SP. Select irradiances of light were administered, and shifts in activity were determined. Photic sensitivity for melatonin suppression was examined in a separate group of animals via pulses of light across a 4 log-unit photon density range, with post-pulse plasma melatonin levels determined via RIA. Phase shifting and melatonin suppression were greater at higher irradiances for both LP and SP. The lower irradiance condition was below threshold for phase shifts in LP but not SP. Melatonin suppression did not vary by photoperiod, and the half saturation constant for fitted sigmoid curves was similar under LP and SP. Thus, the photoperiodic modulation of light sensitivity for phase shifting is conserved across two hamster genera. The dissociation of photoperiod effects on photic phase shifting and melatonin suppression suggests that the modulation of sensitivity occurs downstream of the common retinal input pathway. Understanding the mechanistic basis for this plasticity may yield therapeutic targets for optimizing light therapy practices.     

A Noradrenergic Mechanism Influences the Circadian Timing System in Rats and Hamsters

Brainstem monoaminergic projections to the suprachiasmatic nucleus (SCN), and to the intergeniculate leaflet (IGL), appear to modulate both photic and non-photic effects on the circadian system. Recent work in this laboratory has concentrated on the role of noradrenaline in the regulation of circadian period and phase. Previously, this lab has shown that chronic administration of the alpha₂ adrenergic agonist, clonidine, to rats maintained in constant light (LL) shortens free-running circadian period and promotes dissociation of rhythmicity, while acute clonidine administration to hamsters produces phase shifts similar to those observed with photic stimuli. These results suggest an interaction between clonidine and photic input on circadian rhythmicity, and so the present study was designed to examine systematically the relationship between chronic clonidine administration and photic input in both rats and hamsters. In DD and low intensity LL, clonidine did not alter free-running circadian wheel-running rhythms of rats, but under moderate to high intensity LL, clonidine significantly reduced the period-lengthening effects of LL. Chronic clonidine administration also altered several aspects of circadian phase in hamsters; phase shifts in response to light pulses of varying intensity at CT 19 were reduced; steady-state entrainment phase under a 24-h light-dark cycle (LD 14:10)was delayed; and synchronization to a 23-h light-dark cycle (LD 13:10) was impaired. Clonidine appeared to have little effect on free-running period of hamsters, but a trend towards dissociation of rhythmicity under LL was observed. These effects may reflect an action of clonidine at the photic input pathways to the circadian system, or directly at the circadian pacemaker, since alpha 2 adrenoceptors have been localized both in the suprachiasmatic nucleus (SCN) and in several of its projection areas. As both clinical and experimental studies suggest that clonidine may have depressogenic properties, chronic administration of clonidine to rodents may provide an animal model of the alterations in circadian rhythmicity seen in human depression.     

Nonphotic phase shifting in female Syrian hamsters: interactions with the estrous cycle

Nonphotic phase shifting of circadian rhythms was examined in female Syrian hamsters. Animals were stimulated at zeitgeber time 4.5 by either placing them in a novel running wheel or by transferring them to a clean home cage. Placement in a clean home cage was more effective than novel wheel treatment in stimulating large (> 1.5 h) phase shifts. Peak phase shifts (ca. 3.5 h) and the percentage of females showing large phase shifts were comparable to those found in male hamsters stimulated with novel wheels. The amount of activity induced by nonphotic stimulation and the amount of phase shifting varied slightly with respect to the 4-day estrous cycle. Animals tended to run less and shift less on the day of estrus. Nonphotic stimulation on proestrus often resulted in a 1-day delay of the estrous cycle reflected in animals' postovulatory vaginal discharge and the expression of sexual receptivity (lordosis). This delay of the estrous cycle was associated with large phase advances and high activity. These results extend the generality of nonphotic phase shifting to females for the first time and raise the possibility that resetting of circadian rhythms can induce changes in the estrous cycle.     

Neuropeptide Y microinjected into the suprachiasmatic region phase shifts circadian rhythms in constant darkness

The geniculohypothalamic tract (GHT) is a projection from the intergeniculate leaflet to the suprachiasmatic nucleus (SCN). The GHT exhibits neuropeptide Y (NPY) immunoreactivity and appears to communicate photic information to the SCN. Microinjection of NPY into the SCN has been found to phase shift circadian rhythms of hamsters housed in constant light in a manner similar to the phase shifts produced by pulses of darkness or triazolam injections. In the present study, NPY was injected into the SCN of Syrian hamsters housed in constant darkness and was found to produce phase shifts similar to those seen in hamsters housed in constant light. Microinjections were not followed by wheel running during the subjective day (the time when NPY microinjections are followed by significant phase advances). These data suggest that NPY produces phase shifts by some mechanism other than by inducing wheel running or by inhibiting the response of SCN neurons to light and supports a role for NPY in nonphotic shifting of the circadian clock.     

Non-parametric photic entrainment of Djungarian hamsters with different rhythmic phenotypes

To investigate the role of non-parametric light effects in entrainment, Djungarian hamsters of two different circadian phenotypes were exposed to skeleton photoperiods, or to light pulses at different circadian times, to compile phase response curves (PRCs). Wild-type (WT) hamsters show daily rhythms of locomotor activity in accord with the ambient light/dark conditions, with activity onset and offset strongly coupled to light-off and light-on, respectively. Hamsters of the delayed activity onset (DAO) phenotype, in contrast, progressively delay their activity onset, whereas activity offset remains coupled to light-on. The present study was performed to better understand the underlying mechanisms of this phenomenon. Hamsters of DAO and WT phenotypes were kept first under standard housing conditions with a 14:10 h light–dark cycle, and then exposed to skeleton photoperiods (one or two 15-min light pulses of 100 lx at the times of the former light–dark and/or dark–light transitions). In a second experiment, hamsters of both phenotypes were transferred to constant darkness and allowed to free-run until the lengths of the active (α) and resting (ρ) periods were equal (α:ρ = 1). At this point, animals were then exposed to light pulses (100 lx, 15 min) at different circadian times (CTs). Phase and period changes were estimated separately for activity onset and offset. When exposed to skeleton-photoperiods with one or two light pulses, the daily activity patterns of DAO and WT hamsters were similar to those obtained under conditions of a complete 14:10 h light–dark cycle. However, in the case of giving only one light pulse at the time of the former light–dark transition, animals temporarily free-ran until activity offset coincided with the light pulse. These results show that photic entrainment of the circadian activity rhythm is attained primarily via non-parametric mechanisms, with the “morning” light pulse being the essential cue. In the second experiment, typical photic PRCs were obtained with phase delays in the first half of the subjective night, phase advances in the second half, and a dead zone during the subjective day. ANOVA indicated no significant differences between WT and DAO animals despite a significantly longer free-running period (tau) in DAO hamsters. Considering the phase shifts induced around CT0 and the different period lengths, it was possible to model the entrainment patterns of both phenotypes. It was shown that light-induced phase shifts of activity offset were sufficient to compensate for the long tau in WT and DAO hamsters, thus enabling a stable entrainment of their activity offsets to be achieved. With respect to activity onsets, phase shifts were sufficient only in WT animals; in DAO hamsters, activity onset showed increasing delays. The results of the present paper clearly demonstrate that, under laboratory conditions, the non-parametric component of light and dark leads to circadian entrainment in Djungarian hamsters. However, a stable entrainment of activity onset can be achieved only if the free-running period does not exceed a certain value. With longer tau values, hamsters reveal a DAO phenotype. Under field conditions, therefore, non-photic cues/zeitgebers must obviously be involved to enable a proper circadian entrainment.     

Re-Entrainment Behavior of Djungarian Hamsters (phodopus sungorus) with Different Rhythmic Phenotype Following Light-Dark Shifts

Djungarian hamsters bred at the authors' institute reveal two distinct circadian phenotypes, the wild-type (WT) and DAO type. The latter is characterized by a delayed activity-onset, probably due to a deficient mechanism for photic entrainment. Experiments with zeitgeber shifts have been performed to gain further insight into the mechanisms underlying this phenomenon. Advancing and delaying phase shifts were produced by a single lengthening or shortening of the dark (D) or light (L) time by 6?h. Motor activity was recorded by passive infrared motion detectors. All WT hamsters re-entrained following various zeitgeber shifts and nearly always in the same direction as the zeitgeber shift. On the other hand, a considerable proportion of the DAO animals failed to re-entrain and showed, instead, diurnal, arrhythmic, or free-running activity patterns. All but one of those hamsters that re-entrained did so by delaying their activity rhythm independently of the direction of the LD shift. Resynchronization occurred faster following a delayed than an advanced shift and also after changes of D rather than L. WT animals tended to re-entrain faster, particularly following a zeitgeber advance (where DAO hamsters re-entrained by an 18-h phase delay instead of a 6-h phase advance). However, the difference between phenotypes was statistically significant only with a shortening of L. To better understand re-entrainment behavior, Type VI phase-response curves (PRCs) were constructed. To do this, both WT and DAO animals were kept under LD conditions, and light pulses (15 min, 100 lux) were applied at different times of the dark span. In WT animals, activity-offset always showed phase advances, whereas activity-onset was phase delayed by light pulses applied during the first half of the dark time and not affected by light pulses applied during the second half. When the light pulse was given at the beginning of D, activity-onset responded more strongly, but light pulses given later in D produced significant changes only in activity-offset. In accord with the delayed activity-onset in DAO hamsters, no or only very weak phase-responses were observed when light pulses were given during the first hours of D. However, the second part of the PRCs was similar to that of WT hamsters, even though it was compressed to an interval of only a few hours and the shifts were smaller. Due to these differences, the first light-on or light-off following an LD shift fell into different phases of the PRC and thus caused different re-entrainment behavior. The results show that it is not only steady-state entrainment that is compromised in DAO hamsters but also their re-entrainment behavior following zeitgeber shifts. (Author correspondence: weinert@zoologie.uni-halle.de)     

Phase shifting by novelty-induced running: activity dose-response curves at different circadian times

This study compared phase shifting after novelty-induced running at different circadian times (CTs). In Experiment 1, hamsters were confined to novel wheels for 3 h, starting at CTs 2, 4, 6, 8, 10 or 22. The largest shifts were found at CTs 2, 4 and 6. At each CT there was a relationship between the number of revolutions during the pulse and the size of phase shift. Maximum shifts were usually observed at each CT when animals ran 5000–9000 revolutions during the pulse. In Experiment 2, hamsters were confined to novel wheels for 1 h, also starting at CTs 2, 4, 6, 8, 10 or 22. Unlike with 3-h pulses, the largest shifts with 1-h pulses occurred at CT 8. In Experiment 3, hamsters were shut into a small nest box after a 1-h pulse at CT 8; phase shifting was unaffected, showing that movement about the home cage after a 1-h pulse had ended was not required for shifting. At CTs 2, 4 and 22, 3-h pulses produced shifts but 1-h pulses did not. Possibly, there are two different mechanisms of nonphotic phase shifting that can be activated by being placed in a novel wheel, but the results can also be explained in terms of a single mechanism. Accepted: 8 August 1997     

Addition of a non-photic component to a light-based mathematical model of the human circadian pacemaker

Mathematical models have become vital to the study of many biological processes in humans due to the complexity of the physiological mechanisms underlying these processes and systems. While our current mathematical representation of the human circadian pacemaker has proven useful in many experimental situations, it uses as input only a direct effect of light on the circadian pacemaker. Although light (a photic stimulus) has been shown to be the primary synchronizer of the circadian pacemaker across a number of species, studies in both animals and humans have confirmed the existence of non-photic effects that also contribute to phase shifting and entrainment. We modified our light-based circadian mathematical model to reflect evidence from these studies that the sleep-wake cycle and/or associated behaviors have a non-photic effect on the circadian pacemaker. In our representation, the sleep-wake cycle and its associated behaviors provides a non-photic drive on the circadian pacemaker that acts both independently and concomitantly with light stimuli. Further experiments are required to validate fully our model and to understand the exact effect of the sleep-wake cycle as a non-photic stimulus for the human circadian pacemaker.     

Lesions of the thalamic intergeniculate leaflet alter hamster circadian rhythms

We have investigated the effects of destruction of the geniculo-hypothalamic tract (GHT) on the circadian system of golden hamsters. In the first experiment, intact hamsters were housed in constant darkness, and phase shifts in running-wheel activity rhythms were assessed following 15-min light pulses administered at circadian time (CT) 12 (defined as the beginning of activity), CT 14, CT 18, and CT 20. Responses to light pulses at the same CTs were then reassessed after GHT lesions. Hamsters with complete lesions showed decreases in phase advances caused by light pulses at CT 18 and CT 20. Phase delays elicited by light at CT 12 and CT 14 were not altered. In a second study, intact and GHT-ablated hamsters housed in constant light received 6-hr dark pulses at various CTs. Hamsters with complete GHT ablation showed smaller advances than controls to dark pulses centered on CT 8-10. After 110 days in constant light, 7 of 10 intact hamsters showed splitting of their activity rhythms into two components, while only 1 of the 8 similarly treated ablated hamsters displayed dissociated activity components. Ablated hamsters had significantly shorter free-running periods during the first 35 days of exposure to constant light than did the intact hamsters. These results demonstrate that destruction of the GHT in the hamster alters phase shifting in response to periods of light or dark, and they indicate a role for the GHT in mediating several photic effects on the circadian system.     

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.     

Re-entrainment behavior of Djungarian hamsters (Phodopus sungorus) with different rhythmic phenotype following light-dark shifts

Djungarian hamsters bred at the authors' institute reveal two distinct circadian phenotypes, the wild-type (WT) and DAO type. The latter is characterized by a delayed activity-onset, probably due to a deficient mechanism for photic entrainment. Experiments with zeitgeber shifts have been performed to gain further insight into the mechanisms underlying this phenomenon. Advancing and delaying phase shifts were produced by a single lengthening or shortening of the dark (D) or light (L) time by 6?h. Motor activity was recorded by passive infrared motion detectors. All WT hamsters re-entrained following various zeitgeber shifts and nearly always in the same direction as the zeitgeber shift. On the other hand, a considerable proportion of the DAO animals failed to re-entrain and showed, instead, diurnal, arrhythmic, or free-running activity patterns. All but one of those hamsters that re-entrained did so by delaying their activity rhythm independently of the direction of the LD shift. Resynchronization occurred faster following a delayed than an advanced shift and also after changes of D rather than L. WT animals tended to re-entrain faster, particularly following a zeitgeber advance (where DAO hamsters re-entrained by an 18-h phase delay instead of a 6-h phase advance). However, the difference between phenotypes was statistically significant only with a shortening of L. To better understand re-entrainment behavior, Type VI phase-response curves (PRCs) were constructed. To do this, both WT and DAO animals were kept under LD conditions, and light pulses (15 min, 100 lux) were applied at different times of the dark span. In WT animals, activity-offset always showed phase advances, whereas activity-onset was phase delayed by light pulses applied during the first half of the dark time and not affected by light pulses applied during the second half. When the light pulse was given at the beginning of D, activity-onset responded more strongly, but light pulses given later in D produced significant changes only in activity-offset. In accord with the delayed activity-onset in DAO hamsters, no or only very weak phase-responses were observed when light pulses were given during the first hours of D. However, the second part of the PRCs was similar to that of WT hamsters, even though it was compressed to an interval of only a few hours and the shifts were smaller. Due to these differences, the first light-on or light-off following an LD shift fell into different phases of the PRC and thus caused different re-entrainment behavior. The results show that it is not only steady-state entrainment that is compromised in DAO hamsters but also their re-entrainment behavior following zeitgeber shifts.     

Cocaine modulates pathways for photic and nonphotic entrainment of the mammalian SCN circadian clock

Cocaine abuse is highly disruptive to circadian physiological and behavioral rhythms. The present study was undertaken to determine whether such effects are manifest through actions on critical photic and nonphotic regulatory pathways in the master circadian clock of the mouse suprachiasmatic nucleus (SCN). Impairment of SCN photic signaling by systemic (intraperitoneal) cocaine injection was evidenced by strong (60%) attenuation of light-induced phase-delay shifts of circadian locomotor activity during the early night. A nonphotic action of cocaine was apparent from its induction of 1-h circadian phase-advance shifts at midday. The serotonin receptor antagonist, metergoline, blocked shifting by 80%, implicating a serotonergic mechanism. Reverse microdialysis perfusion of the SCN with cocaine at midday induced 3.7 h phase-advance shifts. Control perfusions with lidocaine and artificial cerebrospinal fluid had little shifting effect. In complementary in vitro experiments, photic-like phase-delay shifts of the SCN circadian neuronal activity rhythm induced by glutamate application to the SCN were completely blocked by cocaine. Cocaine treatment of SCN slices alone at subjective midday, but not the subjective night, induced 3-h phase-advance shifts. Lidocaine had no shifting effect. Cocaine-induced phase shifts were completely blocked by metergoline, but not by the dopamine receptor antagonist, fluphenazine. Finally, pretreatment of SCN slices for 2 h with a low concentration of serotonin agonist (to block subsequent serotonergic phase resetting) abolished cocaine-induced phase shifts at subjective midday. These results reveal multiple effects of cocaine on adult circadian clock regulation that are registered within the SCN and involve enhanced serotonergic transmission.     

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.