Nonphotic Phase Shifting Research Articles

Naloxone’s Effect on Activity-Induced Phase Shifts of Circadian Rhythms

Vigorous running in a novel exercise wheel by hamsters during the subjective day results in large (about 3 hr) phase advances of their circadian rhythms. We administered the opiate receptor antagonist naloxone at 10 mg/kg and 20 mg/kg (i.p.) and found that at 20 mg/kg it significantly reduced phase shifts. This dose of naloxone also reduced the amount of running shown by hamsters in the novel wheel. At the 20 mg/kg dose, the reduced amount of running resulted in smaller phase shifts. The results are consistent with the hypothesis that nonphotic phase shifts are linked to exercise and arousal in hamsters. However, because high amounts of running were accompanied by phase shifting in animals whose opiate receptors were blocked, the results provide no evidence that the rewarding nature of running in rodents is an important causal factor in nonphotic phase shifting beyond its role in promoting running.

Phenotype and function of raphe projections to the suprachiasmatic nucleus

The circadian clock, located in the suprachiasmatic nucleus (SCN), receives a major afferent from the median raphe nucleus (MRN). In the Syrian hamster, only about 50% of the cells giving rise to this afferent contain serotonin. There is mixed evidence as to whether the serotonergic portion of this projection is involved in non-photic phase shifting of circadian locomotor rhythms. In order to better characterize the non-serotonergic projections, we conducted retrograde tract tracing using the beta subunit of cholera toxin combined with multi-label immunohistochemistry. Similar to previous findings, almost half of the retrogradely labeled cells contained serotonin. Additionally, approximately 30% of the retrogradely labeled cells contained vesicular glutamate transporter 3 (VGLUT3), but not serotonin. Surprisingly, some dorsal raphe cholera toxin labeling was also noted, particularly in animals with central-SCN injections. To determine if the non-serotonergic projections were important for non-photic phase shifts elicited by MRN stimulation, the MRN was electrically stimulated in animals pretreated with SCN injection of either the serotonin neurotoxin 5,7-dihydroxytryptamine or vehicle control. Intact animals phase advanced to midday electrical stimulation of the raphe while lesioned animals did not. Together, these results show that although some of the non-serotonergic raphe projections to the SCN contain VGLUT3, it is the serotonergic raphe innervation of the SCN that is critical for non-photic phase shifting elicited by MRN stimulation.

Non-photic phase shifting of the circadian clock: role of the extracellular signal-responsive kinases I/II/mitogen-activated protein kinase pathway

The master circadian clock, located in the suprachiasmatic nucleus (SCN), is synchronized to the external world primarily through exposure to light. A second class of stimuli based on arousal or activity can also reset the hamster circadian clock in a manner distinct from light. The mechanism underlying these non-photic phase shifts is unknown, although suppression of canonical clock genes and immediate early genes has been implicated. Recently, suppression of one of the mitogen-activated protein kinases (MAPK), namely extracellular signal-responsive kinases I/II (ERK), has been implicated in phase shifts to dark pulses, a stimulus with both photic and non-photic components. We investigated the involvement of the ERK/MAPK pathway in phase shifts in response to 3 h of sleep deprivation initiated at mid-day. About three-quarters of animals subjected to this procedure demonstrated large phase advances of about 3 h. Those that shifted exhibited a significant decrease in phosphorylated ERK (p-ERK) in the SCN. Those animals that were perfused during the sleep deprivation also exhibited immunoreactivity for p-ERK in a distinct portion of the ventrolateral SCN. Finally, injections of U0126 to the SCN to prevent phosphorylation of ERK significantly decreased levels of p-ERK but did not produce phase shifts. These data demonstrate that a purely non-photic manipulation is able to alter the activity of the MAPK pathway in the SCN, with downregulation in the SCN shell and activation in a portion of the SCN core.

Illuminating serotonergic gateways for strong resetting of the mammalian circadian clock

circadian clocks regulate animal behavior and physiology by generating daily rhythms and coordinating these with local time. If you sleep well and abstain from flying around the globe or working rotating shift schedules, this pervasive influence is easy to take for granted, a background rhythm of

Short‐term constant light potentiation of large‐magnitude circadian phase shifts induced by 8‐OH‐DPAT: effects on serotonin receptors and gene expression in the hamster suprachiasmatic nucleus

Nonphotic phase-shifting of mammalian circadian rhythms is thought to be mediated in part by serotonin (5-HT) acting in the suprachiasmatic nucleus (SCN) circadian clock. Previously we showed that brief (1-3 days) exposure to constant light (LL) greatly potentiates nonphotic phase-shifting induced by the 5-HT agonist, (+/-)2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT). Here we investigated potential mechanisms for this action of LL, including 5-HT receptor upregulation and SCN clock gene and neuropeptide gene expression. Autoradiographic analysis of ritanserin inhibition of [3H]8-OH-DPAT binding indicated that LL (approximately 2 days) did not affect 5-HT7 receptor binding in the SCN or dorsal raphe. Measurement of 5-HT1A autoreceptors in the median raphe and 5-HT1B receptors in the SCN also showed no effect of LL. In experiment 2, hamsters held under a 14-h light : 10-h dark photocycle (LD) or exposed to LL for approximately 2 days received an intraperitoneal injection of 8-OH-DPAT or vehicle at zeitgeber time (ZT) 6 or 0 and were killed after 2 h of dark exposure. 8-OH-DPAT suppressed SCN Per1 and Per2 mRNAs at both ZTs, as assessed by in situ hybridization. Per1 mRNA was also suppressed by LL alone. In addition, in situ hybridization of arginine vasopressin (AVP) mRNA and vasoactive intestinal polypeptide mRNA showed that LL significantly suppressed the former but not the latter. The LL-induced suppression of SCN Per1 mRNA and AVP mRNA may be involved in LL-induced potentiation of pacemaker resetting, especially as these data provide additional evidence that LL suppresses circadian pacemaker amplitude, thus rendering the clock more susceptible to phase-shifting stimuli.

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.

Opioid induced non-photic phase shifts of hamster circadian activity rhythms

The phase of the circadian pacemaker in hamsters can be shifted by the application of certain non-photic stimuli late in the subjective day. A projection from the intergeniculate leaflet of the thalamus to the circadian pacemaker in the suprachiasmatic nucleus is believed to mediate some types of non-photic phase-shifting stimuli. In hamsters, this projection is immunoreactive to both Neuropeptide Y and enkephalin. Previous work in other laboratories has shown that Neuropeptide Y administration is capable of phase shifting circadian rhythms without the application of light. The present study was undertaken to determine if enkephalinergic compounds likewise have the ability to non-photically phase shift hamster activity rhythms. Hamsters were maintained under conditions of constant darkness and circadian wheel running activity was recorded. Agonists and antagonists selective for κ, μ, and δ opioid receptors were systemically applied without light to hamsters at circadian times 8 and 10 to determine if they were able to elicit phase shifts in wheel running activity rhythms. Of the compounds tested, only the delta opioid agonist BW373U86 significantly affected circadian phase. BW373U86 phase advanced hamster wheel running activity rhythms by approximately 45 min, although total activity levels following drug application were not significantly affected. Changes in the amount of wheel running activity were detected after administration of some μ and κ opioids, although the circadian phase was not altered. These results indicate that enkephalin-mimetic δ opioid agonists are capable of producing non-photic phase shifts in hamster activity rhythms, and that opioids can independently affect circadian phase and activity levels in hamsters.

Nonphotic phase-shifting in Clock mutant mice

Nonphotic phase-shifting was studied in mice bearing the Clock mutation. First, free-running mice heterozygous for Clock and wild-type mice were induced to become active through a 4-h confinement to a novel running over 3 days. Second, mice exposed to light–dark cycle received daily hypocaloric food during 2 weeks, before being transferred to constant darkness and fed ad libitum. Behavioral activation during the mid-subjective day induced 40-min phase advances in the locomotor activity rhythm of wild-type mice, whereas it produced 50-min phase delays in the circadian behavior of Clock/+ mice. Calorie restriction phase-advanced by 80 min the locomotor activity rhythm in wild-type mice, but not in Clock/+ mice. Therefore, the response of the Clock/+ mice to nonphotic phase shifting differs from that of wild-type mice.

Critical Assessment of Methods and Concepts in Nonphotic Phase Shifting

This paper concerns methodological limitations in research on nonphotic resetting of circadian rhythms. There are problems in producing phase responses curves for arousing activity-inducing stimuli when locomotor activity is also used as a phase marker. It is also difficult to define the nature of these nonphotic inputs. Dose-response curves relating amount of wheel running to phase shifts have been overinterpreted, and the measurement of phase shifts is complicated by concomitant changes in period. Some of these points also apply sometimes to chronobiological experiments with light.

Nonphotic phase-shifting and the motivation to run: cold exposure reexamined.

Circadian rhythms in rodents can be phase shifted by appropriately timed activity. This may be dependent on motivational context; running induced by a novel wheel is effective, whereas running induced by cold has been inferred to be ineffective. This issue was reexamined using a different cold exposure procedure. On the first day of constant dark, 6 h before usual dark onset, Syrian hamsters were exposed to cold (+/- 4 degrees C) in their home cages, or were confined to novel wheels for 3 h. Activity rhythms were significantly phase advanced by 92 +/- 10 min following cold exposure and 86 +/- 17 min following novel wheel running, compared to 13 +/- 18 min in a control condition. Most hamsters exhibited eating, drinking, and modest levels of wheel running (1367 +/- 292 counts/6 h) during and for 3 h after cold exposure. Phase shifts following cold were not affected by food and water deprivation but were significantly attenuated by locking the wheel for 6 h beginning at cold onset (24 +/- 12 min). These data indicate that cold-induced running, even at modest levels, is an effective nonphotic Zeitgeber and do not provide support for a hypothesis that motivational contexts determine the phase-shifting value of physical activity.

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 ...

Blocking the phase-shifting effect of neuropeptide Y with light.

Previous studies have indicated that the neuropeptide Y input from the intergeniculate leaflet of the lateral geniculate nucleus to the suprachiasmatic nucleus is the final part of a non-photic phase shifting pathway to pacemakers in hamsters, or that neuropeptide Y is necessary for other pathways to be effective. Experiments in which two stimuli are presented during the same circadian cycle have shown that phase shifts in response to at least two non-photic stimuli are attenuated by a subsequent light pulse during the subjective day. This study was conducted to investigate the neural site of the blocking effect of light on non-photic stimuli. Experiment 1 showed that phase shifts in response to induced wheel-running during the subjective day are greatly attenuated by a subsequent light pulse. Experiment 2 showed that phase shifts in response to injections of neuropeptide Y in the middle of the subjective day were also greatly reduced by a subsequent light pulse. These results provide some insight about the site of the blocking action of light on non-photic phase shifts. Because there is evidence indicating that neuropeptide Y may mediate phase shifts in response to induced activity, and because light was able to block phase shifts produced by neuropeptide Y, we conclude that, in blocking activity-induced shifts, light must act downstream from the release of neuropeptide Y into the suprachiasmatic nucleus.

A serotonin neurotoxin attenuates the phase-shifting effects of triazolam on the circadian clock in hamsters

Several lines of evidence suggest the potential involvement of serotonergic pathways in mediating the effects of activity-inducing stimuli on the circadian clock in rodents. The aim of the present 3 experiments was to examine the effects of the serotonergic neurotoxin, p-hloroamphetamine (PCA, 10 mg/kg) on: (1) the monoamine levels of the hypothalamus, frontal cortex and hippocampus in the hamster; (2) the phase shifts in the circadian rhythm of locomotor activity of hamsters in response to treatment with the short-acting benzodiazepine, triazolam (7.5 mg/kg); and (3) the magnitude of the acute increase in locomotor activity associated with triazolam administration in this species. The administration of PCA to hamsters caused changes of specific monoaminergic systems in the hypothalamus, that were limited to a selective decrease in serotonin levels 7 days post-treatment. The phase shifts of the circadian clock in response to triazolam treatment at CT 6 were considerably attenuated following the administration of the 5-HT neurotoxin. The total amount and the profiles of triazolam-induced wheel-running and general cage activity between CT 6 and CT 12 were not significantly affected by the PCA treatment. The finding that a 5-HT neurotoxin can attenuate the phase-shifting effects of triazolam in hamsters, without interfering with its activity-inducing properties, suggests that serotonergic afferents might be involved in the mechanism for non-photic phase-shifting of the circadian system.

Intergeniculate leaflet lesions and behaviorally-induced shifts of circadian rhythms

The aim of this work was to assess the effect of lesions of the intergeniculate leaflet on nonphotic phase shifts produced by confining hamsters to novel running wheels for 3 h in the middle of the subjective day. In intact hamsters this procedure produces large phase advances provided that the hamsters maintain high levels of wheel running during the confinement. Intergeniculate leaflet lesions blocked or reduced phase shifts after confinement to a novel wheel. However, for most animals these lesions also reduced both the amount of activity during the 3 h pulse in the novel wheel and the amount of daily wheel running in the home cage. To boost activity of lesioned hamsters to levels associated with large phase shifts, the animals were confined to novel wheels at low ambient temperature. The lesioned hamsters still failed to show large phase shifts. The benzodiazepine triazolam also failed to induce phase shifts in lesioned animals, but it induced less activity in lesioned animals as compared to sham-operated controls. The data support the hypothesis that the intergeniculate leaflet conveys information about nonphotic phase-shifting to the circadian pacemaker in the suprachiasmatic nucleus. They also raise the possibility that some effects of intergeniculate leaflet lesions previously interpreted as having a photic basis, might be due to the activity-lowering effect of the lesions.

Nonphotic phase shifting in the old and the cold.

When confined to novel running wheels or when given injections of triazolam in their home cages, old hamsters do not become as active as young hamsters. Therefore, lack of nonphotic phase shifting following such manipulations may stem from insufficient activity or arousal. Phase advances can be obtained in some 10-month-old animals when wheel running during the pulse is increased by the presence of females in estrous condition and in most 18-month-old hamsters by combining confinement to a novel wheel with triazolam injections. These data suggest that there is relatively little if anything wrong in aging hamsters with the nonphotic phase-shifting mechanism itself. The reason why in certain situations old hamsters do not shift appears to be because the nonphotic inputs to these shifting mechanisms are not strong enough. However, when running in novel wheels is increased by carrying out the tests at cold temperatures, most old animals did not show subsequent phase shifts. Evidently it is not running per se that is critical for phase shifts, but probably the motivational context for such running.