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.     

Loss of dexras1 alters nonphotic circadian phase shifts and reveals a role for the intergeniculate leaflet (IGL) in gene-targeted mice

Loss of Dexras1 in gene-targeted mice impairs circadian entrainment to light cycles and produces complex changes to phase-dependent resetting responses (phase shifts) to light. The authors now describe greatly enhanced and phase-specific nonphotic responses induced by arousal in dexras1(-/-) mice. In constant conditions, mutant mice exhibited significant arousal-induced phase shifts throughout the subjective day. Unusual phase advances in the late subjective night were also produced when arousal has little effect in mice. Bilateral lesions of the intergeniculate leaflet (IGL) completely eliminated both the nonphotic as well as the light-induced phase shifts of circadian locomotor rhythms during the subjective day, but had no effect on nighttime phase shifts. The expression of FOS-like protein in the suprachiasmatic nucleus (SCN) was not affected by either photic or nonphotic stimulation in the subjective day in either genotype. Therefore, the loss of Dexras1 (1) enhances nonphotic phase shifts in a phase-dependent manner, and (2) demonstrates that the IGL in mice is a primary mediator of circadian phase-resetting responses to both photic and nonphotic events during the subjective day, but plays a different functional role in the subjective night. Furthermore, (3) the change in FOS level does not appear to be a critical step in the entrainment pathways for either light or arousal during the subjective day. The cumulative evidence suggests that Dexras1 regulates multiple photic and nonphotic signal-transduction pathways, thereby playing an essential role modulating species-specific characteristics of circadian entrainment.     

Nonphotic phase shifting in hamster clock mutants.

Golden hamsters with the tau mutation were kept in the dark and induced to become active through confinement to a novel running wheel for 3 hr. The response of the mutants to this nonphotic phase-shifting stimulus differed from that of wild-type hamsters. The mutants showed larger phase shifts, and their phase response curves differed in shape, with an advance portion at about circadian time 24, a phase at which wild types show delays. The results establish that the tau mutation, in addition to its already known effects, alters the response of the circadian system to nonphotic events.     

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.     

Loss of dexras1 Alters Nonphotic Circadian Phase Shifts and Reveals a Role for the Intergeniculate Leaflet (IGL) in Gene-Targeted Mice

Loss of Dexras1 in gene-targeted mice impairs circadian entrainment to light cycles and produces complex changes to phase-dependent resetting responses (phase shifts) to light. The authors now describe greatly enhanced and phase-specific nonphotic responses induced by arousal in dexras1^?/? mice. In constant conditions, mutant mice exhibited significant arousal-induced phase shifts throughout the subjective day. Unusual phase advances in the late subjective night were also produced when arousal has little effect in mice. Bilateral lesions of the intergeniculate leaflet (IGL) completely eliminated both the nonphotic as well as the light-induced phase shifts of circadian locomotor rhythms during the subjective day, but had no effect on nighttime phase shifts. The expression of FOS-like protein in the suprachiasmatic nucleus (SCN) was not affected by either photic or nonphotic stimulation in the subjective day in either genotype. Therefore, the loss of Dexras1 (1) enhances nonphotic phase shifts in a phase-dependent manner, and (2) demonstrates that the IGL in mice is a primary mediator of circadian phase-resetting responses to both photic and nonphotic events during the subjective day, but plays a different functional role in the subjective night. Furthermore, (3) the change in FOS level does not appear to be a critical step in the entrainment pathways for either light or arousal during the subjective day. The cumulative evidence suggests that Dexras1 regulates multiple photic and nonphotic signal-transduction pathways, thereby playing an essential role modulating species-specific characteristics of circadian entrainment. (Author correspondence: ralph@psych.utoronto.ca)     

Methods of Measuring Phase Shifts: Why I Continue to Use an Aschoff Type II Procedure Despite the Skepticism of Referees

Figure 1 shows the test procedure used in many experiments from this laboratory on nonphotic clock resetting. The animal, a hamster, is in an LD cycle until the day when the stimulus is given. Very close to the time the stimulus is introduced the lights are turned off and remain off until the end of the test, usually just a few days later. This is a modification of Aschoff's (1) type II method for determining phase shifts and phase response curves (PRCs).     

Methods of Measuring Phase Shifts: Why I Continue to Use an Aschoff Type II Procedure Despite the Skepticism of Referees

Figure 1 shows the test procedure used in many experiments from this laboratory on nonphotic clock resetting. The animal, a hamster, is in an LD cycle until the day when the stimulus is given. Very close to the time the stimulus is introduced the lights are turned off and remain off until the end of the test, usually just a few days later. This is a modification of Aschoff's (1) type II method for determining phase shifts and phase response curves (PRCs).     

Nonphotic entrainment in a diurnal mammal, the European ground squirrel (Spermophilus citellus).

Entrainment by nonphotic, activity-inducing stimuli has been investigated in detail in nocturnal rodents, but little is known about nonphotic entrainment in diurnal animals. Comparative studies would offer the opportunity to distinguish between two possibilities. (1) If nonphotic phase shifts depend on the phase of the activity cycle, the phase response curve (PRC) should be about 180 degrees out of phase in nocturnal and diurnal mammals. (2) If nonphotic phase shifts depend on the phase of the pacemaker, the two PRCs should be in phase. We used the diurnal European ground squirrel (Spermophilus citellus) in a nonphotic entrainment experiment to distinguish between the two possibilities. Ten European ground squirrels were kept under dim red light (<1 lux) and 20 +/- 1 degrees C. During the entrainment phase of the experiment, the animals were confined every 23.5 h (T) to a running wheel for 3 h. The circadian rhythms of 6 squirrels entrained, 2 continued to free run, and 2 possibly entrained but displayed arrhythmicity during the experiment. In a second experiment, a photic pulse was used in a similar protocol. Five out of 9 squirrels entrained, 1 did not entrain, and 3 yielded ambiguous results. During stable entrainment, the phase-advancing nonphotic pulses coincided with the end of the subjective day, while phase-advancing light pulses coincided with the start of the subjective day: mean psi(nonphotic) = 11.4 h; mean psi(photic) = 0.9 h (psi defined as the difference between the onset of activity and the start of the pulse). The data for nonphotic entrainment correspond well with those from similar experiments with nocturnal Syrian hamsters where psi(nonphotic) varied from 8.09 to 11.34 h. This indicates that the circadian phase response to a nonphotic activity-inducing stimulus depends on the phase of the pacemaker rather than on the phase of the activity cycle.     

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.     

Differential responses of circadian activity onset and offset following GABA-ergic and opioid receptor activation

The circadian pacemaker in the mammalian suprachiasmatic nuclei is responsive to photic and nonphotic stimuli. In the present study, the authors have investigated the response of activity onset and offset to application of nonphotic stimuli: the benzodiazepine midazolam and the opioid receptor agonist fentanyl. In correspondence with previous studies, both stimuli induced phase advances of the activity onset when given in the mid- to late subjective day. In contrast, activity offset did not phase advance following these injections. Injections during the early subjective day induced small phase delays of the activity onset, while large phase delays occurred in activity offset. Phase shifts, induced at both circadian time zones, were paralleled by an increase in the length of daily activity (alpha). The increase in a remained present during several days after the injection. The different kinetics in phase shifting of the activity onset and offset indicate complexity in phase-shifting behavior of the circadian pacemaker in response to nonphotic stimuli. Moreover, the data show responsiveness of the circadian system to GABA-ergic and opioid receptor activation, not only during the mid- to late subjective day but also during the early subjective day. The data implicate that the early subjective day is an interesting phase for analysis of molecular and biochemical processes involved in nonphotic phase shifting.     

Photic and nonphotic inputs to the diurnal circadian clock

Diurnal animals occupy a different temporal niche from nocturnal animals and are consequently exposed to different amounts of light as well as different dangers. Accordingly, some variation exists in the way that diurnal animals synchronize their internal circadian clock to match the external 24-hour daily cycle. First, though the brain mechanisms underlying photic entrainment are very similar among species with different daily activity patterns, there is evidence that diurnal animals are less sensitive to photic stimuli compared to nocturnal animals. Second, stimuli other than light that synchronize rhythms (i.e. nonphotic stimuli) can also entrain and phase shift daily rhythms. Some of the rules that govern nonphotic entrainment in nocturnal animals as well as the brain mechanisms that control nonphotic influences on rhythms do not appear to apply to diurnal animals, however. Some evidence supports the idea that arousal or activity plays an important role in entraining rhythms in diurnal animals, either during the light (active) or dark (inactive) phases, though no consistent pattern is seen. GABAergic stimulation induces phase shifts during the subjective day in both diurnal and nocturnal animals. In diurnal Arvicanthis niloticus (Nile grass rats), SCN GABA_A receptor activation at this time results in phase delays while in nocturnal animals phase advances are induced. It appears that the effect of GABA at this circadian phase results from the inhibition of period gene expression in both diurnal and nocturnal animals. Nonetheless, the resulting phase shifts are in opposite directions. It is not known what stimuli or behaviours ultimately induce changes in GABA activity in the SCN that result in alterations of circadian phase in diurnal grass rats. Taken together, studies such as these suggest that it may be problematic to apply the principles governing nocturnal nonphotic entrainment and its underlying mechanisms to diurnal species including humans.     

τ Changes After Single Nonphotic Events

Changes in the free-running period of the circadian rhythms of hamsters occur after single nonphotic events such as a 3-h pulse of running induced by being put in a novel wheel. These changes are mostly in the direction of longer periods, and can exceed 0.2 h; the magnitude of the effect depends on the circadian phase of the pulse. The phase response curves for period changes do not match up with those for phase shifts of the rhythms. Data on free-running rhythms after anisomycin injections and after novelty-induced wheel running in τ mutant hamsters support the view that period changes and phase shifts can occur independently of one another.     

Exogenous melatonin reduces the resynchronization time after phase shifts of a nonphotic zeitgeber in the house sparrow (Passer domesticus)

Continuous melatonin administration via silastic implants accelerates the resynchronization of the circadian locomotor activity rhythm in house sparrows (Passer domesticus) after exposure to phase shifts of a weak light-dark cycle. Constant melatonin might induce this effect either by increasing the sensitivity of the visual system to a light zeitgeber or by reducing the degree of self-sustainment of the circadian pacemaker. To distinguish between these two possible mechanisms, two groups of house sparrows, one carrying melatonin implants and the other empty implants, were kept in constant dim light and subjected to advance and delay shifts of a 12-h feeding phase. The resynchronization times of their circadian feeding rhythm following the phase shifts were significantly shorter when the birds carried melatonin implants than when they carried empty implants. In a second experiment, melatonin-implanted and control birds were released into food ad libitum conditions 2 days after either a delay or an advance phase shift. The number of hours by which the activity rhythms had been shifted on the second day in food ad libitum conditions was assessed. Melatonin-implanted house sparrows had significantly larger phase shifts in their circadian feeding rhythm than control birds. This is in accordance with the first experiment since a larger phase shift at a given time reflects accelerated resynchronization. Additionally, the second experiment also excludes any possible masking effects of the nonphotic zeitgeber. In conclusion, constant melatonin accelerates resynchronization even after phase shifts of a nonphotic zeitgeber, indicating that constant high levels of melatonin can reduce the degree of self-sustainment of the circadian pacemaker independent of any effects on the photoreceptive 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.     

Cycle of period gene expression in a diurnal mammal (Spermophilus tridecemlineatus): implications for nonphotic phase shifting.

Ground squirrels, Spermophilus tridecemlineatus, were kept in a 12:12 h light-dark cycle. As expected for a diurnal species, their locomotor activity occurred almost entirely in the daytime. Expression of mPer1 and mPer2 in the suprachiasmatic nucleus was studied at six time points by in situ hybridization. For both these genes, mRNA was highest in the first part of the subjective day (about zeitgeber time 5). This is close to the time when mPer1 and mPer2 expression is maximal in nocturnal rodents. These results have implications for understanding nonphotic phase response curves in diurnal species and thereby for guiding research on nonphotic phase shifting in people.     

Photic and nonphotic circadian phase resetting in a diurnal primate, the common marmoset

Despite the considerable literature on circadian entrainment, there is little information on this subject in diurnal mammals. Contributing to this lack of understanding is the problem of separating photic from nonphotic (behavioral) phase-resetting events in diurnal species. In the present study, photic phase resetting was obtained in diurnal common marmosets held under constant dim light (DimDim; <0.5 lx) by using a 20-s pulse of bright light to minimize time available for behavioral arousal. This stimulus elicited phase advances at circadian time (CT) 18-22 and phase delays at CT9-12. Daily presentation of these 20-s pulses produced entrainment with a phase angle of approximately 11 h (0 h = activity onset). Nonphotic phase resetting was obtained under DimDim with the use of a 1-h-induced activity pulse, consisting of intermittent cage agitation and water sprinkling, delivered in total darkness to minimize photic effects. This stimulus caused phase delays at CT20-24, and entrainment to a scheduled daily regimen of these pulses occurred with a phase angle of approximately 0 h. These results indicate that photic and nonphotic phase-response curves (PRCs) of marmosets are similar to those of nocturnal rodents and that nonphotic PRCs are keyed to the phase of the suprachiasmatic nucleus pacemaker, not to the phase of the activity-rest cycle.     

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.     

Behavioral inhibition of circadian responses to light.

Circadian locomotor rhythms in rodents may be synchronized by either photic or nonphotic events that produce phase shifts of the rhythm. Little is known, however, about how these two types of stimuli interact to produce entrainment. The well-characterized circadian photic response of the golden hamster was examined in situations where a short light pulse and locomotor activity, a nonphotic event, occurred simultaneously. Light-induced phase advances were attenuated when animals were active during light exposure. The results show that circadian responses to light depend upon the environmental situation in which the light is given, and call into question the implicit assumption in circadian rhythm research that phase shifting and entrainment to light-dark cycles depend simply on photic activation of well-known retinofugal pathways. Moreover, since light therapy is becoming an important component in the treatment of circadian-based disorders in humans, the results emphasize the need for evaluation of the behavioral aspects of light therapy protocols.     

Nonphotic entrainment in humans?

Although light is accepted as the dominant zeitgeber for entrainment of the human circadian system, there is evidence that nonphotic stimuli may play a role. This review critically assesses the current evidence in support of nonphotic entrainment in humans. Studies involving manipulations of sleep-wake schedules, exercise, mealtimes, and social stimuli are re-examined, bearing in mind the fact that the human circadian clock is sensitive to very dim light and has a free-running period very close to 24 h. Because of light confounds, the study of totally blind subjects with free-running circadian rhythms represents the ideal model to investigate the effects of nonphotic stimuli on circadian phase and period. Strong support for nonphotic entrainment in humans has already come from the study of a few blind subjects with entrained circadian rhythms. However, in these studies the nonphotic stimulus(i) responsible was not identified. The effect of appropriately timed exercise or exogenous melatonin represents the best proof to date of an effect of nonphotic stimuli on human circadian timing. Phase-response curves for both exercise and melatonin have been constructed. Given the powerful effect of feeding as a circadian zeitgeber in various nonhuman species, studies of meal timing are recommended. In conclusion, the available evidence indicates that it remains worthwhile to continue to study nonphotic effects on human circadian timing to identify treatment strategies for shift workers and transmeridian travelers as well as for the blind and possibly the elderly.     

Late-night presentation of an auditory stimulus phase delays human circadian rhythms

Although light is considered the primary entrainer of circadian rhythms in humans, nonphotic stimuli, including exercise and melatonin also phase shift the biological clock. Furthermore, in birds and nonhuman mammals, auditory stimuli are effective zeitgebers. This study investigated whether a nonphotic auditory stimulus phase shifts human circadian rhythms. Ten subjects (5 men and 5 women, ages 18-72, mean age +/- SD, 44.7 +/- 21.4 yr) completed two 4-day laboratory sessions in constant dim light (<20 lux). They received two consecutive presentations of either a 2-h auditory or control stimulus from 0100 to 0300 on the second and third nights (presentation order of the stimulus and control was counterbalanced). Core body temperature (CBT) was collected and stored in 2-min bins throughout the study and salivary melatonin was obtained every 30 min from 1900 to 2330 on the baseline and poststimulus/postcontrol nights. Circadian phase of dim light melatonin onset (DLMO) and of CBT minimum, before and after auditory or control presentation was assessed. The auditory stimulus produced significantly larger phase delays of the circadian melatonin (mean +/- SD, -0.89 +/- 0.40 h vs. -0.27 +/- 0.16 h) and CBT (-1.16 +/- 0.69 h vs. -0.44 +/- 0.27 h) rhythms than the control. Phase changes for the two circadian rhythms also positively correlated, indicating direct effects on the biological clock. In addition, the auditory stimulus significantly decreased fatigue compared with the control. This study is the first demonstration of an auditory stimulus phase-shifting circadian rhythms in humans, with shifts similar in size and direction to those of other nonphotic stimuli presented during the early subjective night. This novel stimulus may be a useful countermeasure to facilitate circadian adaptation after transmeridian travel or shift work.