Circadian Rhythm Of Wheel-running Activity Research Articles

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.

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.

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.

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.

Melatonin and light induce phase shifts of circadian activity rhythms in the C3H/HeN mouse.

This study examines the effect of light pulses and administration of the pineal hormone melatonin on the circadian activity rhythm of C3H/HeN mice. Mice were housed in constant dark in cages equipped with running wheels. Phase shifts in the circadian rhythm of wheel-running activity were measured following treatment with a 15-min pulse of light (300 lux) or administration of vehicle (ethanol/saline) or melatonin (90 micrograms, sc). Light treatment induced phase changes in circadian activity rhythms; specifically, delays during early subjective night (circadian time [CT] 12.5 to CT 18.5) and advances during late subjective night (CT 0.5). A single dose of melatonin administered at various CTs had no consistent effect on free-running circadian activity rhythms. By contrast, melatonin administration for 3 consecutive days at the same clock time induced advances in circadian activity rhythms by more than 1 h when the first dose was administered at CT 10 and induced delays in circadian activity rhythms by up to 1 h when the first dose was administered between CT 24 and CT 2. With the caveat that multi- ple melatonin treatments are required to induce phase shifts, the results suggest that the circadian timing system controlling the rhythm of wheel-running activity in the C3H/HeN mouse is responsive to both light and melatonin.

Altered circadian rhythmicity in the Wocko mouse, a hyperactive transgenic mutant.

Induced changes in the level of daily activity can alter the period of the mammalian circadian clock. In this report, we examined the period of the circadian rhythm of wheel-running activity in a transgenic neurological mouse mutant, Wocko. Wocko mice display a dominant behavioral phenotype that consists of hyperactivity, circling and head tossing. The period of the circadian rhythm of wheel-running activity in constant dark conditions was significantly shorter in mice expressing the Wocko mutation than in their normal littermates. Total activity, monitored by the interruption of an array of infrared beams, was significantly elevated in Wocko mice. These findings support the view that spontaneous exercise can modulate the circadian timekeeping system.

Effect of a nitric oxide synthase inhibitor, N-nitro- l-arginine methylester, on light-induced phase delay of circadian rhythm of wheel-running activity in golden hamsters

Under constant darkness hamsters demonstrate free-running activity rhythms and light exposure during the early subjective night results in permanent phase delays of the activity rhythm. Recently, we reported that application of glutamate receptor agonists such as N-methyl- d-aspartate could reset the phase of the circadian rhythm of suprachiasmatic nucleus firing activity in vitro via nitric oxide production. In order to confirm this result by in vivo experiment, we examined the effect of nitric oxide synthesis inhibitor on the light-induced phase delay of circadian rhythms of wheel-running activity in hamsters. In vehicle-treated animals, light stimulation at circadian time 13.5 resulted in stable phase delays (1.3 ± 0.63 h), whereas pre-treatment with 150 mg/kg of N-nitro- l-arginine methylester ( l-NAME) significantly attenuated light-induced phase delays (0.72 ± 0.18 h). l-NAME administration alone without light exposure, did not cause phase changes. The l-NAME-induced attenuating effect was reversed by co-administration of l-arginine (300 mg/kg). The present results suggest that nitric oxide production is involved in the light-induced phase delay of the hamster's circadian system.

Effect of chronic tryptophan depletion on the circadian rhythm of wheel-running activity in rats

The effect of chronic treatment with a tryptophan (TRP)-free diet on the free-running circadian wheel-running rhythm and the central serotonergic system was investigated in blinded male rats. The long-term TRP-free diet did not change periods of activity, but disordered their patterns. This seemed to be due to masking, entrainment, enhancement of the morning activity, and obscuring of the activity onset as well as appearance of some periodic activities within the subjective night. A long-term TRP-free diet decreased the concentration of TRP, 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in all brain regions tested: frontal cortex, hippocampus, thalamus, hypothalamus, midbrain, and pons. Density of 5-HT 1A receptor binding was significantly decreased in the frontal cortex and hypothalamus, whereas no significant change was observed in the density of 5-HT 2 receptor binding in all regions. These results suggest that the period of primary circadian pacemaker is not affected, but its oscillation, as well as the coupling strength between the primary and secondary pacemakers, is weakened by the dysfunction of the serotonergic system caused by chronic TRP depletion.

Effects of the 5-HT 1a receptor agonist 8-OH-DPAT and other non-photic stimuli on the circadian rhythm of wheel-running activity in hamsters under different constant conditions

Our findings show that administration of the 5-HT 1A receptor agonist 8-OH-DPAT (8-hydroxy-2[di- n-propylamino] tetralin) was able to induce significant phase-advances 4 h before onset of hamster locomotor activity under constant darkness. All other non-photic treatments applied such as melatonin, dimethyl sulphoxide, Ringer, saline or enforced wheel-running failed to induce any significant phase-advances. Similar results were obtained in pinealectomized animals. In constant light (LL), all treatments produced phase-advances. These results show that: (1) LL seems to be an inappropriate constant condition to study the chronobiological effect of drugs or other non-photic stimuli; (2) the endogenous circadian pacemaker (SCN) can be affected specifically by 8-OH-DPAT.

Circadian wheel running activity rhythms in two strains of domestic rabbit

Results from experiments on two strains of domestic rabbit show that despite their hopping gait, circadian locomotor rhythms can be successfully measured using running wheels specially designed for large laboratory mammals. Three female rabbits of an English outbred strain were monitored in a 13:11 light:dark (LD) cycle and in constant light (LL), while 12 Dutch-Himalayan rabbits were monitored in a 14:10 LD cycle and constant dark (DD). Much stronger rhythms were observed in the latter strain, which under LD conditions showed individuals with patterns that varied from nocturnal ( n = 5), diurnal ( n = 4), crepuscular ( n = 1), and predominantly arrhythmic ( n = 2). In DD, 10 rabbits showed strong free-running rhythms, five with tau (τ) less than 24 h and five longer. The predominantly arrhythmic activity pattern in the remaining two made estimation of τ difficult. Differences in circadian patterns both under LD and DD were unrelated to sex. Results show that locomotor rhythms of the rabbit can be measured using the running wheel, the traditional apparatus of chronobiology. However, the experiments suggest also that the large-bodied breed of rabbits that tend towards obesity, such as the English outbred strain, are relatively inactive and that smaller, more active breeds such as the Dutch-Himalayan may be more suitable for studying circadian rhythmicity if the running wheel is employed.

Phase-shifting mechanisms in the mammalian circadian system: new light on the carbachol paradox

A variety of evidence now suggests that excitatory amino acid receptors mediate the effects of light on the circadian system of mammals. However, the ACh agonist carbachol is the only agent that has been reported to "mimic" the phase-shifting effects of light in vivo. Because the other published evidence for the involvement of ACh in light-mediated phase shifts is weak, we have referred to this situation as "the carbachol paradox." In the present study, we found that the administration of NMDA receptor antagonists could prevent carbachol-induced phase shifts of the circadian rhythm of wheel-running activity recorded from the hamster. In addition, we found that carbachol-induced phase shifts, unlike those produced by light, are not accompanied by induction of Fos-like immunoreactivity in the suprachiasmatic nucleus (SCN). Our data are simply explained by the assumption that the intraventricular administration of carbachol causes phase shifts through a pathway distinct from that of light. Alternatively, if carbachol is acting via the light input pathway, then it must do so by a mechanism independent of Fos induction in the SCN. In either case, elucidating the mechanisms by which carbachol acts in the circadian system may provide novel insights into the cellular events by which phase shifts are generated.

NMDA as well as non-NMDA receptor antagonists can prevent the phase-shifting effects of light on the circadian system of the golden hamster.

The present experiments were designed to evaluate whether the intraventricular administration of excitatory amino acid (EAA) receptor antagonists would prevent light-induced phase shifts of the circadian rhythm of wheel-running activity in the hamster. Administration of the non-N-methyl-D-aspartate (non-NMDA) antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) blocked light-induced phase advances and delays. Similarly, administration of the competitive NMDA receptor antagonist, 3(2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP), prevented light-induced phase advances and delays. Neither drug by itself caused any consistent effect on the phase of the rhythm. These data provide further evidence that EAA receptors mediate the effects of light on the circadian system, and suggest that both NMDA and non-NMDA receptor types may be involved.

An in vitro circadian rhythm of protein synthesis in the rat suprachiasmatic nucleus under tissue culture conditions

Because inhibitors of protein synthesis produce changes in the circadian rhythm of wheel-running activity in rodents, we examined the circadian changes of in vitro protein synthesis by the rat suprachiasmatic nucleus (SCN). We demonstrated a robust circadian rhythm of [ 14C]leucine incorporation as well as [ 3H]2-deoxyglucose (2DG) uptake by the SCN. The peak time of 2DG uptake was around circadian time 6–9 h (CT6-CT9). In contrast to 2DG uptake, the maximum rate of leucine incorporation occurred around CT22-CT0. Thus, the leucine incorporation rate preceded 2DG uptake by 6–9 h. Leucine incorporation was inhibited by protein synthesis inhibitors, but not by tetrodotoxin. Since a robust circadian rhythm of leucine incorporation by the SCN was detected in vitro by using our method, this procedure may be useful to study the circadian clock function of the SCN.

Effects of 5-HT 1A receptor agonists on the circadian rhythm of wheel-running activity in hamsters

The effects of 5-HT 1A receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), buspirone and ipsapirone on wheel-running activity in hamsters were investigated in comparison with those of GABA A receptor agonist muscimol and benzodiazepine triazolam. Intraperitoneal administration of 8-OH-DPAT, buspirone, ipsapirone, muscimol and triazolam at circadian time (CT) 8 (CT 12; onset of activity) induced a significant phase advance of wheel-running activity under constant light conditions. However, administration of these drugs at other CT points did not induce phase changes. The administration of trifluoromethylphenylpiperazine (TFMPP), a 5-HT 1B receptor agonist, at CT8 produced a small phase advance. The phase advance induced by 8-OH-DPAT was blocked by pretreatment with (−)-pindolol, a 5-HT 1A receptor antagonist. In addition, 8-OH-DPAT, buspirone and SM3997 accelerated the rate of re-entrainment to an 8-h phase advance in the light-dark cycle. These observations suggest that 5-HT 1A receptors in the brain participate in the regulation of the circadian rhythm of wheel-running activity in hamsters.

Restricted feeding entrains circadian wheel-running activity rhythms of the kowari

The effect of daily restricted feeding (RF) on the circadian wheel-running rhythms of the kowari, Dasyuroides byrnei, was examined in two experiments. Kowaris were presented with a preferred food (determined in a pilot study) during a daily 2-h meal in the light period of a 14:10 light-dark (LD) cycle (expts 1 and 2), during constant dark (DD) immediately after termination of the LD cycle (expt 1), and during DD when kowaris were free running (expt 1). Results showed that 1) RF elicited anticipatory activity similar in duration and phasing to that observed in the rat; 2) cycles of meal-associated activity free ran for up to 6 days after the termination of RF; 3) activity persists at a phase near that of the former mealtime during periods of food deprivation; and 4) activity indicative of beating between two pacemakers occurred when feeding was restricted to the L period of LD cycles. Together these observations suggest that the activity rhythms of the kowari may be controlled by separate, but possibly coupled, light-entrainable and food-entrainable pacemakers, as are those of the rat.

NMDA receptor antagonists block the effects of light on circadian behavior in the mouse

We report here the results of experiments designed to evaluate whether NMDA receptors mediate the phase shifting effects of light on the circadian rhythm of wheel-running activity in mice. Intraperitoneal administration of either the non-competitive NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine maleate (MK-801), or the competitive NMDA receptor antagonist, 3(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) blocked light-induced phase advances and delays. Neither drug, by itself, caused any consistent effect on the phase of the rhythm. Furthermore, there was no significant difference between the effects of MK-801 on light-induced phase shifts in a retinally degenerate and retinally normal strain of C57 mouse. These data, coupled with previous findings, indicate that excitatory amino acid receptors play an important role in the transmission of light information from the retina to the circadian system.

Sensitivity and integration in a visual pathway for circadian entrainment in the hamster (Mesocricetus auratus).

1. Light-induced phase shifts of the circadian rhythm of wheel-running activity were used to measure the photic sensitivity of a circadian pacemaker and the visual pathway that conveys light information to it in the golden hamster (Mesocricetus auratus). The sensitivity to stimulus irradiance and duration was assessed by measuring the magnitude of phase-shift responses to photic stimuli of different irradiance and duration. The visual sensitivity was also measured at three different phases of the circadian rhythm. 2. The stimulus-response curves measured at different circadian phases suggest that the maximum phase-shift is the only aspect of visual responsivity to change as a function of the circadian day. The half-saturation constants (sigma) for the stimulus-response curves are not significantly different over the three circadian phases tested. The photic sensitivity to irradiance (1/sigma) appears to remain constant over the circadian day. 3. The hamster circadian pacemaker and the photoreceptive system that subserves it are more sensitive to the irradiance of longer-duration stimuli than to irradiance of briefer stimuli. The system is maximally sensitive to the irradiance of stimuli of 300 s and longer in duration. A quantitative model is presented to explain the changes that occur in the stimulus-response curves as a function of photic stimulus duration. 4. The threshold for photic stimulation of the hamster circadian pacemaker is also quite high. The threshold irradiance (the minimum irradiance necessary to induce statistically significant responses) is approximately 10(11) photons cm-2 s-1 for optimal stimulus durations. This threshold is equivalent to a luminance at the cornea of 0.1 cd m-2. 5. We also measured the sensitivity of this visual pathway to the total number of photons in a stimulus. This system is maximally sensitive to photons in stimuli between 30 and 3600 s in duration. The maximum quantum efficiency of photic integration occurs in 300 s stimuli. 6. These results suggest that the visual pathways that convey light information to the mammalian circadian pacemaker possess several unique characteristics. These pathways are relatively insensitive to light irradiance and also integrate light inputs over relatively long durations. This visual system, therefore, possesses an optimal sensitivity of 'tuning' to total photons delivered in stimuli of several minutes in duration. Together these characteristics may make this visual system unresponsive to environmental 'noise' that would interfere with the entrainment of circadian rhythms to light-dark cycles.

Location of the suprachiasmatic nucleus grafts in rats which restored circadian rhythmicity after transplantation

In Wistar male rats whose circadian wheel running activity rhythms were disrupted by bilateral suprachiasmatic nuclei (SCN) lesions, transplantation of neonatal rat SCN into the 3rd ventricle was performed. Out of 49 rats from which adequate wheel running activity records were obtained, 15 rats showed restoration of circadian rhythm starting 2 to 13 weeks (average 1 month) after transplantation. The existence of active SCN neurons in the graft was shown by immunocytochemical reactivity to vasopressin- and VIP-like substances. In all rats, effective grafts were found in the diencephalon, mostly on the wall of the 3rd ventricle.

Do NMDA receptors mediate the effects of light on circadian behavior?

We report here the results of experiments designed to evaluate whether a specific NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine maleate (MK-801), blocks the phase shifting effects of light on the circadian rhythm of wheel-running activity in golden hamsters. Intraperitoneal administration of (+)-MK-801 produced a dose-dependent blockade of both light-induced phase advances and delays. The effect was stereoselective and treatment with related compounds, phenylcyclidine and ketamine, also blocked light-induced phase shifts. MK-801, by itself, did not cause any consistent effect on the phase of the rhythm. These data, coupled with previous findings, indicate that excitatory amino acid receptors play an important role in the transmission of light information from the retina to the circadian system.