Wheel-running Activity Rhythms Research Articles

Fentanyl, a μ-opioid receptor agonist, phase shifts the hamster circadian pacemaker

The phase-shifting effects of the μ-opioid receptor agonist fentanyl on the circadian timing system were investigated in the hamster. Fentanyl injections during the mid-subjective day induced phase advances of the hamsters’ wheel-running activity rhythm. The shifts were not accompanied by an increase in locomotor activity but instead a decrease of activity was often observed. A dose–response curve indicated that with increasing dosage, the response probability increased, while the magnitude of the induced shift remained stable. The present data suggest that there is some role for opioid regulation of the circadian system.

SNC 80, a delta-opioid agonist, elicits phase advances in hamster circadian activity rhythms.

Non-photic stimuli administered to hamsters during the subjective day can cause phase advances in circadian wheel running activity. It is believed that afferent projections from the intergeniculate leaflet of the thalamus to circadian pacemaker cells within the suprachiasmatic nucleus mediate the phase shifting effects of some non-photic stimuli. In hamsters, many of the intergeniculate leaflet afferents contain enkephalin, yet the role of opioids in producing non-photic phase shifts in hamsters has not been reported. In the present study, we show that SNC 80, an agonist for the delta opioid receptor subtype, will phase advance hamster wheel running activity rhythms when administered late in the subjective day. These results indicate that opioids may be involved in modulating the circadian pacemaker in hamsters.

Pituitary adenylyl cyclase-activating peptide: a pivotal modulator of glutamatergic regulation of the suprachiasmatic circadian clock.

The circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus organizes behavioral rhythms, such as the sleep-wake cycle, on a near 24-h time base and synchronizes them to environmental day and night. Light information is transmitted to the SCN by direct retinal projections via the retinohypothalamic tract (RHT). Both glutamate (Glu) and pituitary adenylyl cyclase-activating peptide (PACAP) are localized within the RHT. Whereas Glu is an established mediator of light entrainment, the role of PACAP is unknown. To understand the functional significance of this colocalization, we assessed the effects of nocturnal Glu and PACAP on phasing of the circadian rhythm of neuronal firing in slices of rat SCN. When coadministered, PACAP blocked the phase advance normally induced by Glu during late night. Surprisingly, blocking PACAP neurotransmission, with either PACAP6-38, a specific PACAP receptor antagonist, or anti-PACAP antibodies, augmented the Glu-induced phase advance. Blocking PACAP in vivo also potentiated the light-induced phase advance of the rhythm of hamster wheel-running activity. Conversely, PACAP enhanced the Glu-induced delay in the early night, whereas PACAP6-38 inhibited it. These results reveal that PACAP is a significant component of the Glu-mediated light-entrainment pathway. When Glu activates the system, PACAP receptor-mediated processes can provide gain control that generates graded phase shifts. The relative strengths of the Glu and PACAP signals together may encode the amplitude of adaptive circadian behavioral responses to the natural range of intensities of nocturnal light.

Functional absence of extraocular photoreception in hamster circadian rhythm entrainment

The mammalian circadian pacemaker is entrainable by light via the retina. The putative role of extraocular light perception was investigated in blinded hamsters. These animals were shaved and exposed to a light-emitting pad for either 30 min or 3 h. The absence of any phase-shifting effects on wheel running activity rhythms indicates that extraocular light perception plays no functional role in photic entrainment of the circadian pacemaker in the hamster.

Phase-Shifting Effects of Single and Multiple Light Pulses in the Golden Hamster

In a preliminary attempt to understand the mechanism of light adaptation in the circadian system, the phase-shifting effects of single and multiple light pulses on the rhythm of running-wheel activity were studied in golden hamsters. The results suggest that the reduction in sensitivity due to light adaptation (i.e., refractoriness to light stimulation) starts immediately (less than 1 min) after photic stimulation and that 40 min of stimulation are enough to saturate the system. Recovery of sensitivity can occur within 24 h or less if the photic stimulation is shorter than approximately 10 to 18 h but can take up to 3 weeks if the photic stimulation is longer. This suggests that, although photic stimulation for 40 min is sufficient to totally inhibit further phase-resetting, the process of light adaptation is fully completed only after 10 to 18 h of stimulation. An important implication of the results of this study is that the traditional concept of the phase response curve as a description of the resetting p roperties of the circadian clock is of limited use if light pulses are presented only at a single arbitrary interval after release of the animals into constant darkness.

Interstrain differences in activity pattern, pineal function, and SCN melatonin receptor density of rats.

We investigated the possibility that strain-dependent differences in the diurnal pattern of wheel running activity rhythms are also reflected in the melatonin profiles. The inbred rat strains ACI/Ztm, BH/Ztm, and LEW/Ztm. LEW were examined for diurnal [12:12-h light-dark (LD)] wheel running activity, urinary 6-sulphatoxymelatonin (aMT6s) excretion, melatonin concentrations of plasma and pineal glands, and melatonin receptor density in the suprachiasmatic nuclei (SCN). ACI rats displayed unimodal activity patterns with a high level of activity, whereas BH and LEW rats showed multimodal activity patterns with ultradian components and reduced activity levels. In contrast, the individual daily profiles of aMT6s excretion and mean melatonin synthesis followed a unimodal time pattern in all three strains, suggesting that different output pathways of the SCN are responsible for the temporal organization of locomotor activity and pineal melatonin synthesis. In addition, melatonin synthesis at night and SCN melatonin receptor density at day were significantly higher in BH and LEW rats than in ACI rats. These results support the hypothesis of a long-term stimulating effect of melatonin on its own receptor density in the SCN.

Rapid Resetting of the Mammalian Circadian Clock

The suprachiasmatic nuclei (SCN) contain the principal circadian clock governing overt daily rhythms of physiology and behavior. The endogenous circadian cycle is entrained to the light/dark via direct glutamatergic retinal afferents to the SCN. To understand the molecular basis of entrainment, it is first necessary to define how rapidly the clock is reset by a light pulse. We used a two-pulse paradigm, in combination with cellular and behavioral analyses of SCN function, to explore the speed of resetting of the circadian oscillator in Syrian hamster and mouse. Analysis of c-fos induction and cAMP response element-binding protein phosphorylation in the retinorecipient SCN demonstrated that the SCN are able to resolve and respond to light pulses presented 1 or 2 hr apart. Analysis of the phase shifts of the circadian wheel-running activity rhythm of hamsters presented with single or double pulses demonstrated that resetting of the oscillator occurred within 2 hr. This was the case for both delaying and advancing phase shifts. Examination of delaying shifts in the mouse showed resetting within 2 hr and in addition showed that resetting is not completed within 1 hr of a light pulse. These results establish the temporal window within which to define the primary molecular mechanisms of circadian resetting in the mammal.

Conditioned stimulus control in the rat circadian system depends on clock resetting during conditioning.

The authors examined the ability of a conditioned stimulus (CS; mild air disturbance) previously paired with an entraining light pulse to reset the circadian pacemaker in rats. Rats were entrained to a single 30-min light stimulus delivered every 25 hr or 24 hr (T cycle). Each daily light presentation was paired with the CS. After at least 20 days of stable entrainment to each of the T cycles, the rats were allowed to free run and were then presented with the CS at circadian time 15. CS-induced phase shifts in wheel-running activity rhythms were taken as evidence for conditioning. For the most part, conditioning occurred after CS-light pairings on the 25-hr but not 24-hr T cycle. The results suggest that CS control of the circadian clock phase depends on the effect that the entraining light pulse has on the clock during conditioning.

Roles of suprachiasmatic nuclei and intergeniculate leaflets in mediating the phase-shifting effects of a serotonergic agonist and their photic modulation during subjective day.

Serotonin (5-HT) has been implicated in the phase adjustment of the circadian system during the subjective day in response to nonphotic stimuli. Two components of the circadian system, the suprachiasmatic nucleus (SCN) (site of the circadian clock) and the intergeniculate leaflet (IGL), receive serotonergic projections from the median raphe nucleus and the dorsal raphe nucleus, respectively. Experiment 1, performed in golden hamsters housed in constant darkness, compared the effects of bilateral microinjections of the 5-HT1A/7 receptor agonist, 8-hydroxydipropylaminotetralin (8-OH-DPAT; 0.5 microgram in 0.2 microliter saline per side), into the IGL or the SCN during the mid-subjective day. Bilateral 8-OH-DPAT injections into either the SCN or the IGL led to significant phase advances of the circadian rhythm of wheel-running activity (p < .001). The phase advances following 8-OH-DPAT injections in the IGL were dose department (p < .001). Because a light pulse administered during the middle of the subjective day can attenuate the phase-resetting effect of a systemic injection of 8-OH-DPAT, Experiment 2 was designed to determine whether light could modulate 5-HT agonist activity at the level of the SCN and/or the IGL. Serotonergic receptor activation within the SCN, followed by a pulse of light (300 lux of white light lasting 30 min), still induced phase advances. In contrast, the effect of serotonergic stimulation within the IGL was blocked by a light pulse. These results indicate that the respective 5-HT projections to the SCN and IGL subserve different functions in the circadian responses to photic and nonphotic stimuli.

Selective MT2 melatonin receptor antagonists block melatonin-mediated phase advances of circadian rhythms.

This study demonstrates the involvement of the MT2 (Mel1b) melatonin receptor in mediating phase advances of circadian activity rhythms by melatonin. In situ hybridization histochemistry with digoxigenin-labeled oligonucleotide probes revealed for the first time the expression of mt1 and MT2 melatonin receptor mRNA within the suprachiasmatic nucleus of the C3H/HeN mouse. Melatonin (0.9 to 30 microg/mouse, s.c.) administration during 3 days at the end of the subjective day (CT 10) to C3H/HeN mice kept in constant dark phase advanced circadian rhythms of wheel running activity in a dose-dependent manner [EC50=0.72 microg/mouse; 0.98+/-0.08 h (n=15) maximal advance at 9 microg/mouse]. Neither the selective MT2 melatonin receptor antagonists 4P-ADOT and 4P-PDOT (90 microg/mouse, s.c.) nor luzindole (300 microg/mouse, s.c.), which shows 25-fold higher affinity for the MT2 than the mt1 subtype, affected the phase of circadian activity rhythms when given alone at CT 10. All three antagonists, however, shifted to the right the dose-response curve to melatonin, as they significantly reduced the phase shifting effects of 0.9 and 3 microg melatonin. This is the first study to demonstrate that melatonin phase advances circadian rhythms by activation of a membrane-bound melatonin receptor and strongly suggests that this effect is mediated through the MT2 melatonin receptor subtype within the circadian timing system. We conclude that the MT2 melatonin receptor subtype is a novel therapeutic target for the development of subtype-selective analogs for the treatment of circadian sleep and mood-related disorders.

An NK 1 receptor antagonist affects the circadian regulation of locomotor activity in golden hamsters

Substance P (SP) is a neuromodulator which may participate in the photic regulation of the circadian timing system in mammals. The biological effects of SP are mediated by interaction with specific receptors, designated as NK 1, NK 2, and NK 3. The NK 1 subtype receptor is expressed in the circadian system. Experiment 1 was designed to test whether an NK 1 antagonist mimics the effects of dark pulses. Hamsters were housed in constant lighting conditions, either constant darkness or constant light (around 250 lx), and they received an i.p. injection of either the specific NK 1 receptor antagonist, L-760,735 (5 mg/kg), or saline during the mid-subjective day, a time when dark pulses cause a phase-advance in circadian rhythm of locomotor activity. After treatment with the NK 1 antagonist, significant phase-advances of wheel-running activity rhythm were found in constant light, but not in constant darkness. Experiment 2 was designed to test the ability of the NK 1 antagonist to block the phase-delaying and/or the phase-advancing effects of light in animals kept in constant darkness. Phase-advances of locomotor activity rhythm that can normally be induced by light pulses given during the late subjective night were markedly reduced by pre-treatment with the NK 1 antagonist. By contrast, phase-delays that can be induced by lights pulses given during the early subjective night were unaffected by the NK 1 antagonist. These data support the hypothesis that SP within the circadian system may, by interacting with NK 1 receptors, modulate photic responses of the SCN pacemaker.

Effects of aging on the circadian rhythm of wheel-running activity in C57BL/6 mice.

The effects of age on the circadian clock system have been extensively studied, mainly in two rodent species, the laboratory rat and the golden hamster. However, less information is available on how aging alters circadian rhythmicity in a commonly studied rodent animal model, the mouse. Therefore, in the present study we compared the rhythm of wheel-running activity in adult (6-9 mo) and old (19-22 mo) C57BL/6J mice maintained under different lighting conditions for a period of 4 mo. During this period, mice were subjected to phase advances and phase delays of the light-dark (LD) cycle and eventually to constant darkness (DD). In LD (12 h light, 12 h dark), old mice exhibited delayed activity onset relative to light offset and an increase in the variability of activity onset compared with adult mice. After a 4-h phase advance of the LD cycle, old mice took significantly longer to reentrain their activity rhythm when compared with adult animals. Old mice also demonstrated a decline in the number of wheel revolutions per day and a tendency toward a decrease in the length of the active phase. An increase in fragmentation of activity across the 24-h day was obvious in aging animals, with bouts of activity being shorter and longer rest periods intervening between them. No age difference was detected in the maximum intensity of wheel-running activity. In DD, the free-running period was significantly longer in old mice compared with adults. In view of the rapidly expanding importance of the laboratory mouse for molecular and genetic studies of the mammalian nervous system, the present results provide a basis at the phenotypic level to begin to apply genetic methods to the analysis of circadian rhythms and aging in mammals.

Circadian rhythm of self-exposure to light in the golden hamster

Golden hamsters were maintained in constant darkness with free access to a running wheel and to a lever that they could press to turn on the lights. The animals exhibited a circadian rhythm of lever pressing (and, therefore, of self-exposure to light) in synchrony with the rhythm of running-wheel activity. Although most light exposure occurred during early subjective night (when single light pulses are known to cause phase delays of circadian rhythms), there was no significant difference between the circadian periods before and after the animals were allowed to self-stimulate. It is suggested that the conservation of circadian period was due to a reduction in photic sensitivity caused by the observed pattern of light exposure (multiple brief pulses rather than single long pulses).

Is energy expenditure in the hamster primarily under homeostatic or circadian control?

1. In order to discriminate between homeostatic and circadian control of energy expenditure, this paper considers whether a shorter circadian cycle will produce a proportional reduction in energy expenditure (so that expenditure per unit time is conserved) or alternatively whether energy expenditure will be compressed into the shorter cycle (so that energy expenditure per cycle is conserved). To answer this question, we measured energy expenditure in tau mutant hamsters (whose free-running circadian period has been reduced to about 20 h by a single gene mutation) and wild-type hamsters (whose free-running circadian period is about 24 h). 2. In one experiment, the circadian rhythm of running-wheel activity of tau mutant hamsters was compared with that of wild-type hamsters. The rate of running was not affected by the mutation and, consequently, the total amount of activity per cycle was significantly less in mutants than in wild-type hamsters, whereas the total amount of activity per unit time was nearly the same. 3. In a second experiment, we measured energy expenditure by indirect calorimetry. Metabolic rate was not affected by the mutation and, consequently, the total amount of energy expended per cycle was significantly less in mutants than in wild-type hamsters but equivalent per unit time. 4. Because the amount of energy expenditure and locomotor activity was found to be proportional to the circadian cycle, we conclude that expenditure per unit time-rather than expenditure per circadian cycle-is conserved in the mutant animals. Therefore, we infer that energy expenditure in hamsters is primarily under homeostatic, not circadian, control. Further research is necessary to determine whether this inference can be applied to other species.

Effect of prolonged fasting and subsequent refeeding on free-running rhythms of temperature and locomotor activity in rats

This study investigated the possible effect(s) of prolonged fasting and subsequent ad lib refeeding on the circadian organization of rats kept in constant darkness. Free-running rhythms of wheel-running activity and body temperature were studied in rats fasted during a 7-day interval followed with ad lib refeeding started either at subjective midday, i.e., CT6 (circadian time 6) or subjective midnight, i.e., CT18. Phase-shifts of temperature acrophases were similar to those of activity acrophases. During fasting, phase-shifts were phase-advanced (1 circadian h on the average) in most cases. During refeeding, they were mostly phase-delays (2 circadian h on the average) independently of the circadian time of refeeding, i.e., ad lib refeeding did not act as a Zeitgeber. In conclusion, prolonged fasting and subsequent refeeding induce opposite effects on the circadian organization.

Ventromedial Nuclei of the Hypothalamus are Involved in the Phase Advance of Temperature and Activity Rhythms in Food-Restricted Rats Fed During Daytime

Daily rhythms are synchronized to the light-dark cycle (LD) via a circadian clock located in the suprachiasmatic nuclei. A timed caloric restriction phase advances daily rhythms of body temperature and wheel-running activity in rats kept under LD. Because lesions of the ventromedial hypothalamic nuclei (VMH) prevent the fasting-induced changes in the day-night pattern of activity, it was hypothesized that the VMH might participate in the caloric restriction-induced phase changes. To test this hypothesis, rats with electrolytic or ibotenic acid lesions of VMH and control rats were fed 2 h after lights on 50% of ad lib food intake. During the preceding fed state, rats with electrolytic lesions of VMH displayed a less marked day-night difference in locomotor activity and a phase-advanced acrophase of temperature rhythm (2 h) compared to those of sham-operated rats. These effects were not found in fed rats with ibotenic lesions of VMH, suggesting that these effects of electrolytic lesions were due to disruption of undetermined fibers of passage. In response to a timed caloric restriction, the nocturnal peak of temperature rhythm was phase advanced by 7 h in sham-operated rats. Their day-night pattern of activity was also phase advanced towards the time of feeding. In both groups of food-restricted VMH-lesioned rats, the acrophase of temperature rhythm plateaued 3 h later than in sham-operated group. The phase advance of body temperature was, therefore, reduced to 4 h by ibotenic lesions of VMH and to 2 h by electrolytic lesions. Except for a feeding-associated component of activity expressed in control and VMH-lesioned rats, no significant change in day-night pattern of activity was detected in VMH-lesioned rats, either by electrolytic or ibotenic lesions. These results indicate that neuronal damage of the VMH limits the phase-advancing properties of a timed caloric restriction on the daily rhythms of temperature and locomotor activity.

Muscarinic receptors mediate carbachol-induced phase shifts of circadian activity rhythms in Syrian hamsters

Carbachol, a non-specific cholinergic agonist, when administered intraventricularly or directly into the suprachiasmatic nucleus (SCN), causes phase-dependent shifts in circadian rhythms of wheel-running activity in rodents. The cholinergic receptor subtype involved in mediating these carbachol-induced phase shifts, however, remains uncertain. In order to investigate this issue we injected carbachol into the SCN through indwelling cannulas at circadian times (CT) 6, 14 and 22 in Syrian hamsters ( Mesocricetus auratus) maintained in constant darkness. Carbachol elicited large phase advances at CT 6 (69.8±15.7 min; mean±S.E.M.) and CT 22 (83.9±24.8 min) and phase delays at CT 14 (59.7±18 min). We attempted to block the carbachol-induced phase shifts at these three phases using specific antagonists of nicotinic and muscarinic receptors. Mecamylamine, a nicotinic receptor antagonist, did not block carbachol-induced phase shifts at any of the phases tested. Atropine, a muscarinic receptor antagonist, blocked carbachol-induced phase shifts at CT 6 (−11.6±4.8 min; Mean phase shift±S.E.M.) and CT 22 (−20±6.6 min), suggesting that carbachol mediates its phase-shifting effects at these phases through muscarinic receptors.

Circadian Phase-Response Curves for Simulated Dawn and Dusk Twilights in Hamsters

Phase shifts of the circadian rhythm of wheel-running activity were compared in Syrian hamsters maintained in constant darkness and exposed to 1-h naturalistic dawn or dusk twilight ramps (0.003–10 1x), or to 1-h rectangular light pulses (1 1x) providing equal photon exposure. The phase-response curves (PRCs) for dusk and rectangular pulses were virtually identical and resembled the PRC for dawn pulses, except that the mean phase advance caused by dawn pulses at circadian time 19 (CT 19) was approximately 1 h smaller. This difference could not be accounted for by differences in the amount of wheel-running observed during light pulse exposure, because the animals ran more during dusk pulses than during either of the other two pulse types. In a second experiment, 15-min rectangular light pulses (1 1x) immediately preceded by a 47-min dawn ramp caused smaller phase delays at CT 13 than rectangular pulses alone, despite a 40% increase in total photon exposure, but phase advances at CT 19 did not differ between the two light treatments. These results indicate that phase shifts of the circadian pacemaker in hamsters are determined primarily, though not entirely, by total photon exposure. They also indicate that dawn pulses may be less effective than dusk or rectangular pulses at certain circadian phases, possibly due to light adaptation during the early portion of the dawn twilight.

Photic entrainment in hamsters: effects of simulated twilights and nest box availability.

Entrainment of wheel-running activity rhythms was compared in hamsters exposed to daily light-dark (LD) cycles with abrupt transitions between 0 and 10 lux or with artificial twilights simulating summer solstice conditions at 41 degrees N latitude but truncated at 10 lux. The photoperiod in LD-rectangular was set at 16.24 h, equating the total light (in lux.min) emitted under the two schedules. The LD cycles were maintained for 35 days and were followed by 14 days of constant darkness (DD). Half the animals in each condition had access to a dark nest box connected to the outer compartment by a tunnel, the remaining animals being confined to a single compartment. Body temperature and locomotor activity inside the nest boxes were recorded by telemetry. Movements between the nest box and the outer compartment were monitored and the data were used to calculate light exposure at different times of the day. In all groups, the phase angle difference between wheel-running onset and dusk was more positive than that between activity offset and dawn. Hamsters with access to nest boxes, however, had later onsets, earlier offsets, and shorter activity durations (alpha s) than those without. These effects could be accounted for by the difference in light exposure between the nest and no-nest animals, particularly light exposure in the morning. The inclusion of twilights also resulted in later onsets and shorter alpha s, but the differences were relatively small and were only observed in the nest animals. The day-to-day variability in activity onset was negatively correlated with onset time and was smaller in the twilight/nest animals than in the other three groups. Most animals showed an expansion of alpha during the first few days of DD, resulting from a rapid advance of activity onsets relative to offsets. The period of the rhythms, determined from the first five activity onsets in DD, was negatively correlated with the balance of evening and morning light exposure. These results are discussed in the context of nonparametric entrainment of compound pacemakers.

Daily restricted feeding effects on the circadian activity rhythms of the stripe-faced dunnart, Sminthopsis macroura.

The effect of daily restricted feeding (RF) on the circadian wheel-running activity rhythms of the stripe-faced dunnart (Sminthopsis macroura) was examined. Dunnarts were presented with a 2-h meal in the middle of the light period of a 14:10 light:dark (LD) cycle and during constant dim light (LL). No meal-anticipatory activity (AA) was observed in any of the dunnarts during the experiment. This contrasts with previous work where AA has been reported in dunnarts subjected to RF. In LL, RF acted as a weak zeitgeber for the circadian activity rhythms of the dunnart. Evidence supporting this observation was the fact that 4/8 dunnarts' activity rhythms were entrained by RF, 2 showed relative coordination, and 1 exhibited bouncing phenomenon. In other species of marsupials and in rats, it has been proposed that RF entrains a food-entrainable pacemaker, which, in turn, entrains, via coupling, the suprachiasmatic-based, light-entrainable pacemaker. The findings of the present study differ from those reported previously in that no observable AA was entrained but the light-entrainable pacemaker was entrained by RF. In the dunnart, it remains to be determined whether RF directly entrains the light-entrainable pacemaker or whether RF entrains a food-entrainable pacemaker and in turn, via coupling, the light-entrainable pacemaker.