INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

CIRCADIAN RHYTHMS ARE SYNCHRONIZED to the day-night cycle by the process of entrainment in which photic stimuli adjust the phase of the endogenous circadian pacemaker until it adapts a stable phase relationship to the photocycle. Resetting of the pacemaker proceeds reliably even if the light-dark (LD) cycle is artificially phase shifted by as much as 12 h or if animals are phase shifted repeatedly during reentrainment (3, 6, 9). The behavior of circadian rhythms during reentrainment has been a useful tool to elucidate some general features of pacemaker function such as the temporal relationship between the pacemaker and overt rhythms, the presence of transient cycles during reentrainment, and the asymmetry of reentrainment rates via phase advances or delays (6, 19). These reentrainment phenomena have been explained at a formal level of analysis by a nonparametric model of entrainment in which light produces abrupt daily phase shifts of the pacemaker (19). The response of the pacemaker to light pulses changes across the circadian cycle, but the basic circadian pattern of responses [i.e., the phase response curve (PRC)] bears strong similarities among a diverse number of species. In fact, the shape of the PRC among diurnal rodents is similar to that of nocturnal rodents (7), even in those that entrain primarily via modulation of pacemaker velocity (18).

Phase shifts of the photocycle are invariably followed by phase changes in the circadian pacemaker. We know of no reports in which organisms failed to reentrain to phase shifts of typical LD cycles (e.g., 8-16 h of light/24 h). Siberian hamsters have, however, proven to be exceptional in this regard. These animals are maintained in 16 h light/day and readily reentrained when the LD cycle was phase advanced or delayed by 1 or 3 h, but they failed to reentrain to phase advances or delays of 5 h (23). Hamsters that failed to reentrain free ran for several months or became arrhythmic despite the continued presence of the LD cycle (25). The most consistent feature of free-running activity rhythms in these "nonentrained" Siberian hamsters was that the period [i.e., tau ()] of their rhythms increased after the phase shift as it does under constant light (LL). We calculated mean s from 24.8 to 25.3 h in separate experiments (25) with animals that free ran after the photocycle was phase delayed by 5 h. These values are similar to those reported for Siberian hamsters monitored in LL under similar light intensities [25.3 h (13), 24.7 h (14), and 24.8 h (21)]. We also observed that, after a few months of free running in the photocycle, activity rhythms in some hamsters eventually degrade and become arrhythmic like those of other nocturnal species maintained in LL for long durations (8, 10, 28). By contrast to LL, long-term exposure to constant darkness (DD) does not degrade circadian organization in Siberian hamsters or other nocturnal rodents. Activity rhythms of nocturnal rodents are generally more robust and free run with shorter s in DD than in LL (4, 5, 19). These observations indicate that the effects of a 5-h phase shift on  and activity rhythm coherence in hamsters are similar to those induced by exposure to LL.

The disruptive effects of long-term bright light exposure on activity rhythms are not permanent and can be reversed by reinstating the LD cycle or transferring animals to DD (8). Even rats that are arrhythmic under LL can regain circadian organization within 1 wk of transfer to DD (10). Body temperature and sleep-wake rhythms that are eliminated by long-term exposure to bright light are restored within a few days of reinstating the LD cycle and then free run when subsequently held in dim light (12). The tendency of DD to ameliorate the disruptive effects of LL on rhythm organization led us to hypothesize that exposure to DD could restore normal entrainment in our nonentrained hamsters. The present study tested whether reentrainment could be restored to Siberian hamsters that failed to reentrain to a phase-delayed photocycle by housing them in DD for a brief period of time before returning them to the LD cycle.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Housing conditions. Siberian hamsters (Phodopus sungorus sungorus) were bred in our colony from original stock provided by Dr. Irving Zucker of the University of California, Berkeley, CA. Animals were maintained three to four per cage from birth in a 16:8-h LD cycle (lights on at 0200 PST) and ambient temperature of 22°C. Housing conditions and illumination specifications in the hamster colony and experiment rooms were as previously described (25). Light fixtures contained two cool white (4,100°K) fluorescent tubes (Philips 40 W); light intensity was 350 lx in the center of the room and 10-45 lx on the cage floors depending on cage location in the room. Light measurements were made with cage lids and water bottles in place and food hoppers filled; the light meter lens (ILM 350, Iso-Tech) pointed upwards from the cage bottom for these measurements. Animals were provided cotton batting for nesting material; food (Purina chow #5015) and tap water were available ad libitum. All procedures were carried out in accordance with the guidelines of the American Physiological Society for animal care and use in research (1).

Activity. Activity was measured by passive infrared motion detectors as described previously (25). Each detector is mounted directly above the tip of the water bottle sipper tube, and the coverage pattern is configured so that activity levels primarily reflect drinking behavior but also include locomotor activity that occurs directly under the sipper tube. The temporal resolution for detecting successive bouts of activity is 1-2 s between bouts. Activity bouts were summed in 10-min intervals and stored on computer.

Data analysis. Activity rhythm periods were determined by a standard deviation-based periodogram analysis (11) of 10-day intervals during entrainment and 5-day intervals during free runs. The shorter intervals during free runs were necessary because  changed noticeably over a 5- to 7-day period at several phases of these experiments. Peaks in the periodogram were deemed statistically significant if they exceeded the 99.9% confidence interval limit. Q values (i.e., power) associated with  were used to compare rhythm coherence as described previously (25). Activity rhythms were considered entrained or free running based on periodogram analysis. Because a 10-min sampling interval was used for data acquisition, periodogram analysis occasionally estimated rhythm period to be 23.83 or 24.17 h (i.e., 24.00 ± 1 h sampling interval) in entrained animals. Rhythms were considered entrained if the period estimate was 23.83, 24.00, or 24.17 h and if daily rhythm onsets maintained a stable phase relationship to the LD cycle. The phase relationship to the LD cycle was considered stable when daily activity onsets occurred within a range of 60 min over 10 successive days. Although day-to-day variability in timing of activity onsets is typically <30 min, this definition allowed for occasional outliers in daily activity onsets. Rhythms were considered free running in the LD cycle if periodogram analysis estimated  to be either <23.83 or >24.17 h.

Experiment protocol. Equal numbers of male and female hamsters were used in all three experiments. Data were combined by gender because no significant sex differences were found in any dependent measure. Animals were separated and housed singly in the same photoperiod as in the animal colony (LD 16:8, lights on at 0200 PST). Fourteen days later, the LD cycle was phase delayed via an extension of the light phase by 5 h (lights on at 0700) and animals were divided into three groups (n = 12 each). The first group, designated DD0, was exposed to DD beginning at the end of the 5-h extension of the light phase and held there for 12 days. The second and third groups, designated DD5 and DD14, were moved to DD at 5 or 14 days, respectively, after the phase shift and kept there for 14 days.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Circadian organization under LD and DD conditions. The mean times of activity onsets were calculated for free-running animals on the day that each group went into DD. The mean times of activity onsets among DD0, DD5, and DD14 animals were 0.3 ± 0.2 and 0.3 ± 0.2 h after dark onset and 0.3 ± 0.4 h after light onset, respectively, and did not differ significantly (ANOVA, P > 0.05). The number of days that elapsed between the phase shift and exposure to DD had a significant impact on the number of animals that reentrained (² = 12.7, P < 0.01). All DD0 hamsters reentrained, but only 42% of DD5 and 42% of DD14 hamsters reentrained to the photocycle (n = 5 of 12 from DD5 and DD14 groups).

View larger version (62K): [in this window] [in a new window]   Fig. 1.   Representative activity records of animals moved to constant darkness (DD) on the day of the phase shift (DD0). Time of dark onset was at zeitgeber time 16 h before the phase shift and after DD. The days on which the 5-h phase delay (PD) occurred, DD onset began (DD), and the light-dark (LD) cycle was reinstated are indicated (). Data are double plotted to facilitate visualization of rhythms. Note that s are >24 h for about the first 5 days in DD and then <24 h for the remainder of DD.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   (A) and  (B) for all animals that free ran in DD. Mean (±SE) s and s for DD0 (filled bars; n = 12), DD5 (open bars; n = 7), and DD14 (gray bars; n = 8) hamsters. Data were obtained during the last 10 days of entrainment before the phase shift (Baseline), the first 5 days after the phase shift (Post-PS), days 1-5 in DD (Early DD), days 9-14 in DD (Late DD), and the last 10 days in the reinstated LD cycle (LD).  is not given for LD2 because not all free-running animals reentrained. None of the arrhythmic animals in which rhythms spontaneously returned in DD were included in these analyses. *P < 0.01 compared within groups to Post-PS phase of experiments (Dunnett's post hoc correction applied). No comparison of s was made between baseline and Post-PS phases of the study because there was no variability in  during baseline.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Representative waveforms before (A) and after (B) the phase shift for an animal moved to DD on day 0 (DD0); mean waveforms are from the last 10 days of data before the phase shift (A) and from days 30 to 40 after the LD cycle was reinstated (B). Mean (±SE) phase angle of entrainment (C) and  (D) for all animals (n = 12) before the phase shift (Baseline) and 30-40 days after the LD cycle was reinstated (LD). Note delayed activity onset is accompanied by extension of active phase into morning hours. See Fig. 4 for explanation of tick labels. *P < 0.001 compared with baseline (paired t-test).

View larger version (63K): [in this window] [in a new window]   Fig. 4.   Representative activity records of animals moved to DD beginning 5 days after the phase shift. Times of dark onsets were at zeitgeber time 8 h before the phase shift and at 13 h after DD. Left: animal is arrhythmic for most of DD and regains rhythms shortly before LD cycle is reinstated; right: hamster failed to reentrain despite a robust circadian rhythm in DD. See Fig. 1 for explanation of symbols.

View larger version (61K): [in this window] [in a new window]   Fig. 5.   Representative activity records of animals moved to DD beginning 14 days after the phase shift. Time of dark onset was at zeitgeber time 16 h before the phase shift and after DD. See Fig. 1 for explanation of symbols.

Predictors of reentrainment. Activity rhythms were compared among animals that did or did not reentrain to determine which parameters of circadian organization best predicted reentrainment. Data from DD5 and DD14 groups were combined for this analysis because these two groups did not differ in the proportion of animals that reentrained, or in their mean s, s, or times of activity onsets during the last 5 days of DD. Animals that were arrhythmic before or during DD were excluded from these analyses. We predicted that reentrainment would be more likely to occur if the LD cycle was reinstated so that the dark phase coincided with an animal's active phase because light exposure during subjective night can prevent entrainment in these animals (25).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Distribution of activity onsets for DD5 and DD14 animals. Individual times of activity onsets on the last day of DD for DD5 () and DD14 () hamsters that reentrained (E) or free ran (FR) when the LD cycle was reinstated on the next day. Bar at top indicates phase of reinstated LD cycle. Mean times of activity onsets did not differ among animals that reentrained compared with those that failed to reentrain (t-test, P = 0.81). Roughly equal numbers of animals that entrained or free ran were active during the dark as well as the light phase of the LD cycle.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Changes in  and  in DD predicted which animals reentrained. Mean (±SE) change in  (A) and  (B) at 3 different phases of the study for animals that reentrained (filled bars) or free ran (open bars) when the photocycle was reinstated.  and  are expressed as the difference between values obtained at the Post-PS, Early DD, and Late DD phases of the study to those obtained during baseline entrainment before the phase shift. Tick labels are defined in Fig. 4. *P < 0.05, **P < 0.001 compared with entrained animals.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Most Siberian hamsters fail to reentrain to a 5-h phase delay of a 16:8-h photoperiod and continue to free run for several months despite the continued presence of the LD cycle (23-25). Because only ~9% of hamsters spontaneously reentrain to a 5-h phase shift, reentrainment rates of 100% for DD0 and 42% for DD5 and DD14 animals indicate a marked effect of DD to promote reentrainment. Because the majority of hamsters free run with stable rhythms after a 5-h phase shift, the failure to reentrain must involve changes either in input pathways to the central pacemaker or within the pacemaker itself. We consider it unlikely that input pathways to the pacemaker were compromised after the phase shift because of the changes in  and  that occurred when the LD cycle was replaced with DD, which indicates that the pacemaker could discriminate LD from DD. Therefore, the inability to spontaneously reentrain to a 5-h phase shift of the photocycle probably reflects changes within the pacemaker itself rather than to its afferent pathways. We cannot rule out, however, the possibility that photoreceptors may also influence reentrainment. Photoreceptors are able to integrate light information over relatively short periods of time (2, 27), thus it remains possible that LD cycle history may also be encoded in photoreceptors.

The efficacy of DD to restore entrainment was greatly influenced by the interval between the phase shift and start of DD. All animals in the DD0 group reentrained and did not experience changes in  while in DD. This suggests that the extra 5 h of light experienced on the day of the phase shift were insufficient to prevent reentrainment. For the DD0 group, DD may have prevented, rather than reversed, changes in entrainment mechanisms as was the case for DD5 and DD14 animals that reentrained. The critical events that ultimately prevent reentrainment appear to occur within 5 days of the end of the light exposure on the day of the phase shift. In a prior study (22), we found that  was maximally compressed 2 days after a 5-h phase delay and, in the present study, we found that maintenance of a compressed  is associated with a failure to reentrain. It seems likely, therefore, that changes in circadian organization that prevented reentrainment to the photocycle in our previous studies were completed within 48 h of the phase shift.

One unexpected finding from the DD0 group suggests a novel flexibility in how  and the PRC interact to produce entrainment. All of the animals in this group reentrained with substantial delays in their times of activity onsets. Such delays are typically indicative of increases in  (19), but s of these hamsters resumed their normal DD values well before the end of DD exposure and one can assume that  was not affected by reinstating the LD cycle. Thus, the delay in activity onsets indicates that the 5-h phase delay altered either the shape of the photic PRC or the phase relationship between the PRC and the animal's active phases. Either of these possibilities is incongruous with documented relationships among , PRC, and the phase relationship between activity and the photocycle for other species (19). Relationships among these variables may be particularly labile in Siberian hamsters. Recently, it has been shown that two light pulses, delivered on sequential nights, made Siberian hamsters arrhythmic even though the animals were housed in a LD cycle; subsequent access to running wheels restored circadian organization to activity (26). Because this species is unique in many of its responses to typical lighting regimens, it provides a novel system for future studies on the relationships among , PRC, and entrainment.

The likelihood of reentrainment in DD5 and DD14 animals could be predicted from changes in  and  during DD exposure but not from the time of activity onset when the photocycle was reinstated. Because our previous studies showed that 5 h of light exposure at night had such a marked effect on rhythm organization (25), we were concerned that reentrainment would be less likely if the LD cycle was reinstated in such a way that the animals' active phases were coincident with the light phase. This hypothesis was not supported. Hamsters that were active during the light phase of the photocycle were equally likely to reentrain as those that were active during the dark. By contrast, changes in  and  that occurred in DD were good predictors of whether animals would reentrain. Although  increased and  decreased after the phase shift in all DD5 and DD14 hamsters, these effects were reversed in DD only in those animals that subsequently reentrained to the photocycle. This expansion of  was complete and stabilized before the end of DD, thus, it is unlikely that longer DD exposure would have increased the number of animals that reentrained.

We examined the interaction between the LD cycle and activity rhythms after the phase shift to determine whether the pattern of light exposure was contributing to the maintenance of the free runs. Siberian hamsters that failed to reentrain generally free ran with s that ranged from 25.0 to 25.5 h and s close to 7.5 h (23, 25). This combination of  and , in the presence of an 8-h dark phase, means that these animals were exposed to light during part of their subjective night on nearly every day of their free run. This pattern of light exposure is important because, in Siberian hamsters, light exposure that is limited to subjective night increases  to values that are similar to those observed in our previous studies of unentrained animals (14) and may have additional effects on entrainment. Therefore, it is possible that disruptions in entrainment were initiated by the phase shift and sustained by the animal's interaction with the photocycle. Hamsters in the DD0 group, for example, were not exposed to light after the phase shift and readily reentrained.

The individual differences that could account for variability in response to DD among the DD5 and DD14 groups remain unclear to us but may be related to the general phenomenon of "photoresponsiveness" of photoperiodic animals. Within populations of photoperiodic species, there are often subsets of animals that fail to respond to the decreases in daylength that signal approach of winter (i.e., nonresponders). Siberian hamster nonresponders generally have longer s, shorter s, and lower PRC amplitudes in short daylengths than do responders (20). The animals in this study that failed to reentrain had s and s in DD that were similar to those of nonresponders, whereas animals that reentrained were more similar to responders. Our colony of hamsters was derived from breeding stock in which approximately one-half of the animals were photoperiodic nonresponders (16, 17). This is intriguing because one-half of the DD5 and DD14 animals maintained short s and long s in DD as one would expect for photoperiodic nonresponders and suggests that our entrainment phenomena are related to photoperiodic responsiveness. Although it is possible that these photoperiodic phenotypes may underlie circadian changes observed in DD, it does not explain why 90% of the entire colony failed to reentrain to a 5-h phase shift of the LD cycle (25). In that situation, photoperiodic responsiveness does not appear to differentiate "reentrainers" from "nonreentrainers." There is an additional important difference between the animals that failed to reentrain in this study and photoperiodic nonresponders.  of Siberian hamsters that are nonresponders in short daylengths increases consistently by several hours when hamsters are housed in DD (15); these animals subsequently undergo gonadal regression (15). If our nonentrainers were also nonresponders,  would have increased in DD, but it did not. Thus, the available evidence does not provide a decisive link between photoperiodic and entrainment phenotypes. Nevertheless, we are currently breeding populations of photoperiodic responders and nonresponders to directly investigate the relationships among these phenotypes.