Physiology&Behavior,Vol.52, pp. 985-995, 1992
0031-9384/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
Printedin the USA.
Resetting of a Feeding-Entrainable Circadian Clock in the Rat F R I E D R I C H K. S T E P H A N
Department of Psychology, Psychobiology-Neuroscience Program, Florida State University, Tallahassee, F L 32306-1051 Received 7 April 1992 STEPHAN, F. K. Resetting of a feeding-entrainable circadian clock in the rat. PHYSIOL BEHAV 52(5) 985-995, 1992.-Reentrainment of anticipatory activity (AA)after phase shifts of food access was studied in rats with lesionsof the suprachiasmatic nuclei. Eight- or 10-h phase delays of feeding time resulted in delaying transients of AA. Twelve-h phase shifts and some 10-h phase advances resulted in rapid reentrainment (2-3 days) without visible transients. Most phase advances of 10 h resulted in delaying transients while 8-h advances induced transients with advancing and delaying components in a number of rats. Split transients were not prevented by advancing mealtime in 1-h steps. Phase shifts of food in multiple steps failed to accelerate delay shifts but retarded advance shifts. After the first 8-h phase delay shift, increased activity reappeared at the preshift phase of AA, simultaneously with anticipation of the phase-shifted meal time. The observation of split transients indicates that two or more circadian oscillatorsmediate entrainmentto mealtime, and the reappearance of AA at a previously establishedphase suggest the possibilitythat this system has a memory of phase displacement. Circadian rhythms
Entrainment
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transients. Finally, a few rats show a combination of delaying transients and simultaneously AA to the mealtime (19). The purpose of the present study was to conduct a more extensive investigation of the resetting of AA in rats with SCN lesions. It should be noted that phase shifts of food access have been studied in intact rats, but the results are influenced by the presence of the light-entrainable circadian pacemaker which retards the reentrainment of AA (20). The emphasis of the present study was to reexamine resetting of AA to 8-h phase advances and delays. Ten-h and 12-h phase shifts were also imposed to cover a broader range of phase displacements. In addition, single step phase shifts were compared with multiple step phase shifts in order to test whether the rate and direction of transients could be controlled. Stepwise phase shifts correspond to a temporary T cycle (T = period of food access), and because the upper limit of entrainment is near 31 h (4,16), it seemed possible that large phase delays could be induced to accelerate reentrainment and that multiple step advances might prevent delaying transients. Finally, the phase stability of AA following reentrainment was assessed by imposing 1-3 days of food deprivation immediately after restricted feeding or after ad lib feeding.
THE ability of rats to anticipate a single meal per day by an increase in activity beginning several hours prior to mealtime was reported almost 70 years ago (9). During the past two decades it has become increasingly clear that this anticipatory activity (AA) reflects the entrainment of a circadian pacemaker that is anatomically and functionally distinct from the circadian pacemaker that entrains to light-dark cycles. First, lesions of the suprachiasmatic nucleus (SCN) which abolish many (but perhaps not all) circadian rhythms in rats maintained in ad lib feeding conditions [for review, see (10)] do not abolish the rise in temperature or serum corticosterone (7) or the development of AA prior to mealtime (4,16,23). Second, the limits of entrainment to mealtime are in the circadian range (22 to 31 h) (4,16), and the upper limit of entrainment to mealtime is considerably longer than for light pulses (18). In addition, the onset of AA relative to food access depends systematically on the period of feeding, thus representing the phase angle of entrainment (2,3). Third, although AA rarely persists into ad lib feeding conditions, it nearly always appears during total food deprivation, even after many weeks of intervening ad lib conditions (5,6,8,11). Fourth, following phase shifts of food access, AA reentrains gradually over several cycles (19), and such transients are characteristic of endogenous oscillators. However, several aspects of reentrainment appear to be different from those observed after phase shifts of LD cycles. In particular, 6- or 8-h phase advances of mealtime induce three modes of reentrainment. Some rats display delaying transients, i.e., the system responds as if the advances were 18- or 16-h delays, respectively. Other rats anticipate the phase-shifted mealtime by the second or third day without clearly discernable
METHOD
Animals, Housing, and Activity Recording Sixteen male adult Sprague-Dawley rats (mean body weight 290 g; SEM + 6.8 g) with SCN lesions were housed individually in sound-attenuated chambers that enclosed an activity wheel with a small adjacent cage. An exhaust fan provided fresh air flow and masking noise. Water was available ad lib throughout the study and food access was controlled by a motorized gate.
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The chambers and the experimental room were maintained in DD. During cleaning (weekly) and restocking of food hoppers (at 3-5-day intervals at irregular times of day) the door of the experimental room was left ajar. The brief exposure to low levels of illumination from an adjacent room had no discernable systematic effect on activity. Wheel revolutions were recorded by means of an infrared light-emitting diode and a phototransistor mounted across a tab on the running wheel. Contact circuits recorded licks and eating. All three measures were monitored continuously by a computer and cumulative counts were stored on disk every 10 min. The motorized food gate was under computer control.
phase shift consisted of a 10-h phase advance in a single step for the first group and in ten 1-h steps for the second group. The first group was then food deprived for 3 days, followed by 8 days of ad lib feeding and 2 additional days of food deprivation (see Figs. 1 and 2). Because the rats in the second group required much longer to reentrain and only three rats produced useful data for the complete experiment, the last food deprivation procedure was not used for this group. RESULTS
Histology and Periodicity Analysis
Rats were anesthetized with sodium pentobarbital (5.2 mg/ 100 g b.wt.) and received 0.05 cc atropine sulfate (IM). Electrolytic lesions were aimed at the SCN. With the incisor bar 5 mm above the interaural line, stereotaxic coordinates were 1.3 mm anterior to bregma, _+0.2 mm lateral, and 9.6 mm ventral to the dura. A 2 mA anodal direct current was passed for 18 s through a stainless steel electrode (0.4 mm diameter) which was insulated except for 0.5 mm at the tip. A total of 26 rats received attempted SCN lesions. Drinking was monitored for 3-4 weeks in standard hanging cages, and 16 rats that had the most disrupted circadian rhythms were selected for the experiment.
Based on light microscopic examination, no SCN tissue was identified in any of the 16 rats. The lesions had been made deliberately large so that portions of the anterior hypothalamic area, medial preoptic area, retrochiasmatic area, and the optic chiasm sustained damage. The success of the lesions was also evaluated by examining the last 12 days of drinking prior to transferring the animals to the activity wheels and the first 8 days of ad lib conditions in the wheels with the chi-square periodogram. None of the animals had significant peaks between 18 and 30 h. Drinking data were used because some rats had very low levels of wheel running in baseline conditions which could have made it difficult to detect residual circadian rhythmicity.
Histology
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Rats were deeply anesthetized with sodium pentobarbital and perfused intracardially with normal saline, followed by 10% formalin. Brains were removed and stored in sucrose formalin for at least 10 days and then embedded in gelatin. Frozen sections were cut in the coronal plane at 40 um through the region of damage. Every fourth section was mounted on slides and stained with cresyl violet for light microscopic examination.
Eight-hour delays. All 8-h delays of food access resulted in delaying transients of anticipatory activity (see Figs. 1-4). (The data from rat M60-2 could not be used because of very low activity levels.) The number of days required to reentrain to the single-step delay was between 4 and 6 days on the first shifts. Rats which were exposed to this procedure on the second or third shift required on the average only 3 days to reentrain AA (range 2-5 days; Figs. 1-4). After the first phase delay, increased activity appeared near the preshift phase position of AA some 2-7 days after the day of the phase shift (Figs. 1 and 2). This persisting activity will be discussed separately below. For twostep delays, no consistent effect on days to reentrainment occurred (range 2-6 days, Figs. 1 and 2, 3rd delay), whereas fourstep delays obviously prevented reentrainment in fewer than 4 days (Fig. 1, 2nd delay; Fig. 2, 1st delay). The event records, as well as the activity pattern averaged over four 26-h days (Fig. 5, top), show that AA of five rats followed the daily 2-h delays. In two rats (M60-4, M60-5), the onset of AA shifted by less than 2 h per day so that the onset of AA preceded mealtime by about 8 h (Fig. 5, top). After the first phase shift, the phase angle of entrainment (onset of AA to mealtime) was reduced from about 5 h to 3 h following reentrainment. This effect is visible in the event records and in the distribution of activity averaged over 6 days preshift and days 11-17 postshift (Fig. 6, top row). The peak level of AA was also reduced in six of seven rats following reentrainment. In part, this effect is due to the absence of AA on some of the days used for averaging. Ten-hour delays. Ten-h delays resulted in delaying transients in all seven rats exposed to this procedure (one rat died prior to this shift). While the onset of AA delayed systematically in all rats, in three rats the activity was split into two bouts on the second day. The second bout began about 3 h prior to the new food access time (see Fig. 3, M61-1; Fig. 4, M61-8). On the average, reentrainment occurred in 4 days (range 3-5 days). Twelve-hour phase shift. Only one of seven rats displayed delaying transients to the 12-h phase shift (Fig. I, M60-4). In the remaining rats, AA persisted at the pre-shift phase for one
Surgery
Data Analysis Event records of wheel running, drinking, and contacts with the food hopper were double plotted (48 h horizontal time scale). Wheel running records were the primary measure in the experiment. For the illustrations, the vertical height was plotted as the square root of the number of revolutions per 10 min. Zero counts were printed as one pin and maximum symbol height was clipped at ->81 responses (9 pins). For selected data segments, the amount of activity was assessed by averaging 30-min values over a number of consecutive days. The effect of SCN lesions on circadian rhythms was assessed with the chi-square periodogram (15). PROCEDURE Animals were kept in ad lib feeding conditions for 8 days. To ease the transition to restricted feeding, food access was limited to 4 h/day (1400-1800) for 8 days. The access time was then reduced to 2 h (1400-1600) for 14 days to establish stable anticipatory activity. Eight rats were then exposed to three consecutive 8-h phase delays of food access, in a single step, in four 2-h steps, or in two 4-h steps (see Figs. 1 and 2). Eight rats were exposed to two consecutive 8-h phase advances, in one 8-h step or in eight 1-h steps. The third phase shift was an 8-h delay (see Figs. 3 and 4). The first group was then exposed to a 12-h phase shift and the second group to a 10-h delay of food access time. Eight days after these phase shifts, all rats were food deprived for 3 days, followed by 10 days ofad lib feeding and then placed on restricted feeding at the previous phase position. The last
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to three days and then vanished while AA to the phase shifted food access appeared in 2 to 4 days (Fig. 1, M60-3; Fig. 2; M605 and M60-7). Persisting activity. A surprising result was the appearance of a band of activity beginning near the preshift phase of AA after the initial 8-h phase delay. This activity emerged 2 to 7 days after the phase shift and sporadically disappeared and reappeared (Figs. 1 and 2). On most days, the activity began near the preshift phase of AA. As shown by the distribution of activity averaged over days 11-17 postshift, the amount of this secondary activity was substantial, and in some cases approached preshift levels (e.g., Fig. 6, top row, dashed lines).
Phase Advances Eight-hour advance. None of the rats exposed to one-step advances displayed daily advancing transients between the old and the new phase. AA to the phase shifted access time appeared within 2-3 days. At this point in most rats, the onset of AA continued to advance over the next 2-3 days (e.g., Fig. 3, M61-
6 shows the clearest indication of advancing transients after the second phase shift) and appeared reentrained between 3-5 days. For four rats exposed to the 8-h advance in l-h steps, AA advanced on a daily basis, indicating entrainment to a 23-h T cycle (e.g., Fig. 3, M61-4, 2nd advance). Four rats displayed AA on the first 2-3 and on the last 2-3 days of the shift but did not anticipate food access for 1-3 days in between (e.g., Fig. 3, M611, 2nd advance). In these rats the duration of AA was very short, indicating that the system was very close to the limits of entrainment. The distribution of activity averaged over 8 consecutive 23-h days clearly shows the presence of AA, although the average level of activity is low for rats which did not display AA every day (Fig. 5, bottom). Both types of phase shift were accompanied by a secondary component of activity in 15 of 16 instances. In five cases, this component delayed until it reached the new phase of food access (e.g., Fig. 3, M61-1, 2nd advance; M61-6, 1st advance; Fig. 4, M61-7, 1st and 2nd advance). Since these rats also displayed advancing transients or AA entrained to a 23-h T-cycle, this
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mode of reentrainment involves splitting of transients. In rat M61-7 (Fig. 4), this activity trails the phase shifted food access and appears to delay a second time. In other rats, unstable bands of activity occurred after the phase shifted meal time (e.g., Fig. 3, M61-4, 1st advance). Ten-hour advance. In response to the 10-h advance in a single step, two of seven rats displayed systematic delaying transients (Fig. 1, M60-3, M60-4, last phase shift). Three rats showed a response similar to that observed after 12-h phase shifts. AA continued for 2 days at the preshift phase and then vanished. AA appeared at the new phase after 2-3 days without intervening transients (e.g., Fig. 2, M60-5). The other two rats showed a single delaying transient on the second day after the shift (e.g., Fig. 2, M60-7). Due to equipment problems, only four rats could be evaluated for the 10-h advance in 1-h steps. One of these entrained to the T = 23-h cycle. This rat was a replacement which had not been exposed to previous phase shifts. A second rat entrained for 6 days but then lost entrainment (Fig. 4, M61-8). One rat displayed delaying transients which continued through the T cycle and then failed to entrain once more after the new phase position of the meal had been established. Entrainment finally occurred when the transients approached meal access for the third time (Fig. 4, M61-7). For 3 days, delaying transients and AA to the phase shifted meal can be seen simultaneously. The fourth rat displayed bouts of activity 3-4 h after the mealtime on 7 of the 10 days of the T cycle (Fig. 3, M61-4).
Phase Stability Following reentrainment after the 12-h phase shift, seven rats were food deprived for 2 days. The onset of AA on the second day had shifted less than 1 h in five rats (e.g., Fig. 1, M60-3, M60-4) but appeared delayed by about 3 h in the other two rats (Fig. 2, M60-5; M60-7). The 10-h delay shift was also followed by 2 days of food deprivation. One rat (M61-1) appeared in poor condition and was sacrificed on the second day. Because food was returned after the onset of AA on the third day, phase stability could be assessed for 3 cycles. Two rats showed successive phase advances (Fig. 3, M61-4; Fig. 4, M61-8), two showed successive delays, and two retained a fairly stable phase position (e.g., Fig. 4, M61-7). Twenty-two days after the 10-h advance in a single step, seven rats were food deprived for 3 days, followed by 8 days of ad lib feeding and 2 additional days of food deprivation to again assess the phase stability of AA. During the first food deprivation segment, AA remained near the established phase, or at least within the variability of AA during restricted feeding in all rats (e.g., Figs. 1 and 2). During the second food deprivation segment, AA had shifted by as much as 8 h from the last entrained phase position (e.g., Figs. 1 and 2, last FD condition). Eating and drinking. The contact eatometers used were not completely reliable for all rats and do not provide quantitative information about food intake. Nevertheless, as far as could be determined, rats contacted the food hopper at all meals, regardless of the presence of absence of AA or the type of transients after phase shifts.
PHASE SHIFTS OF FEEDING TIME
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Event records of drinking show that many rats displayed anticipatory licking in addition to anticipatory wheel running. Furthermore, the pattern of secondary activity components as well as split transients was also reflected in drinking. This indicates that these responses are not peculiar to wheel running but reflect a more general activation of behavior. Representative examples are shown in Fig. 7. Bodyweight. At perfusion, the mean body weight (N = 14) was 367 g, SEM _+ 14.3 g. All rats had gained weight during the 156 days of manipulation of food access. DISCUSSION The present results confirm and extend previous observations regarding reentrainment of AA in rats with SCN lesions (19).
Phase delays of feeding time by 8 or 10 h induce delaying transients and rapid reentrainment of AA. For single-step phase delays, reentrainment was faster on the second and third shift than on the first shift. While this effect could not be assessed statistically because of the small N, an earlier study dealing with the limits of entrainment suggests a strong history dependence of the feeding-entrainable pacemaker, i.e., the upper limit, was at least 31 h when T was changed sequentially from 24 to 27, 29, and 31 h, whereas no rat entrained AA when T was abruptly changed from 24 to 31 h (16). The strong tendency of this pacemaker to delay was also confirmed since 8- or 10-h phase advances induced delaying transients in some rats. Eight-h phase delays in two or four steps had little effect on the rate of reentrainment, except to prevent reentrainment in
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FIG. 7. Double-plotted segments of event records of drinking for two rats exposed to 8-h delays (top) or 8-h advances (bottom) of food access. Compare the close correspondence of the temporal pattern of drinking with that of wheel running shown in Figs. 1 and 3. Dashed lines indicate potential splitting of transients for M60-3.
994 fewer than 4 days when 2 h steps were used. These results suggest that this pacemaker rapidly slows down when mealtime is delayed. Consequently, reentrainment takes place in a few days and cannot be accelerated by presenting the Zeitgeber at intermediate phases. The rate of reentrainment to single step 8-h or 10-h delays is surprisingly rapid, especially after the first delay shift, requiring only 4 days or less in 15 of 16 instances. Unusual modes of reentrainment were again observed with 8-h phase advances (19) and also with 10-h advances and 12-h phase shifts. One mode involved reentrainment without apparent intervening transients, particularly during the 10-h advances and 12-h phase shifts. Here, activity continued at or near the preshift phase for 1 or 2 days in a fashion similar to that of rats placed in total food deprivation (4,24). By the second or third day, the phase-shifted meal time was preceded by AA. This could be the result of strong resetting of the pacemaker (25) or alternatively, entrainment of a second oscillator at the new phase position and decoupling of activity from the oscillator at the previous phase position. A two-oscillator interpretation is supported by reentrainment consisting of split transients, which were particularly common during 8-h phase advances. In these cases, delaying transients could be observed concomitantly with advancing transients or apparent entrainment to food access (23-h T cycle). Since one oscillator cannot generate two different periods simultaneously, it seems most parsimonious to assume that two oscillators become temporarily uncoupled when the phase shifted Zeitgeber falls on a critical phase. The possibility that the feeding entrainable pacemaker consists of more than one oscillator is also supported by the ability of rats to anticipate two meals with different periods (e.g., 24 h and 24.5 h) per circadian cycle (17,21). Although few animals in this study showed activity after food access, termed succeeding activity (3), in a few cases one component of split transients appeared to arise from succeeding activity (e.g., Fig. 4, M61-7, 1st phase shift). This raises the possibility that a second oscillator, entrained at a lagging phase, is responsible for succeeding activity. The observation of AA during food deprivation revealed a considerable instability of phase after apparent resetting of the pacemaker. Following the 12-h phase shift and reentrainment, AA during food deprivation was either unshifted or delayed. This group of rats had only experienced phase delays before. The other group had been exposed to two-phase advances and a 10-h delay prior to food deprivation, and several rats showed advances in AA during food deprivation. This suggests that the phase shift history might influence the phase stability of the system in the absence of periodic cues. During the last food deprivation segment, rats expressed bouts of activity which could not readily be extrapolated to the previous entrained phase of AA. Either the pacemaker free ran through ad lib feeding at periods considerably longer or shorter than 24 h or the bouts were triggered by a second oscillator at a different phase position. In all rats, the onset of this activity was between 2300 h (Fig. l, M60-4) and 0600 h (Fig. 2, M60-5, M60-7), so that it is highly unlikely that it was triggered by external cues. An unexpected finding was the reappearance of activity near the preshift phase of AA after the first phase delay shifts. One explanation might be that these rats responded to an exogenous laboratory cue, because it is known that food-deprived rats become more sensitive to external disturbances (l, 14). This interpretation was used to account for recent observations that AA during food deprivation probes appeared near the last entrained phase when rats with SCN lesions were fed in the early afternoon,
STEPHAN but less consistently so when rats were fed at night (12). However, the authors point out that not all their findings are consistent with the external cue hypothesis and provide alternative explanations. In the present study, two factors argue against this interpretation. First, this activity did not appear every day and was also expressed on weekends, when potential noise cues are drastically reduced. Second, the onset of this activity was variable within as well as between animals. The only noise audible to human observers in the experimental room was from activity wheels. Consequently, the external cue hypothesis would require that animals respond to unspecified cues while ignoring audible cues from other rats. On the other hand, systematic advances and delays in the onset of these activity bouts over several cycles may indicate an underlying periodic process (see Fig. 7). Consequently, a tentative interpretation is that this activity is mediated by a second circadian oscillator, i.e., the initial phase delay shifts also may induce splitting, as was the case with 8-h phase advances. If a second oscillator is involved in generating this persisting activity, it becomes necessary to consider a mechanism which would permit the apparent return to a previous phase position, i.e., after the first phase shift the onset of the persisting activity was on the average near that of AA prior to the phase shift. Since it seems likely that the phase shifted Zeitgeber alters the phase (and period) of both oscillators, either a memory of previous phase or more likely of the amount of phase displacement would seem to be required to allow an oscillator to return to a prior phase position. Such a mechanism would have adaptive significance in that ifa food source disappeared from a usual time of day, a memory of time when food was previously available would be advantageous. Phase memory has been postulated previously, based on observations with intact rats which had been entrained to restricted feeding, followed by ad lib feeding and constant light or by a phase shift of the LD cycle. During food deprivation probes, AA emerged at a constant phase relationship to the free-running or to the phase-shifted activity rhythm (8,11). However, these results could also be accounted for by coupling between the feeding- and light-entrainable pacemakers which could preserve their phase relationship [see (20)]. In the present study, the light entrainable pacemaker is absent and, consequently, cannot serve as a phase reference. Since not all circadian rhythms may be abolished by SCN lesions (13), one possibility is that another circadian oscillator which is unaffected by periodic feeding could serve as an intemal phase reference. However, this study provides no direct information on this hypothesis. In summary, the results of the present study are consistent with the hypothesis that the feeding entrainable pacemaker (like the light-entrainable pacemaker) consists of more than one oscillator. The most compelling evidence for this interpretation is the occurrence of split transients, especially after 8-h phase advances, and possibly after 8-h delays of the Zeitgeber. A rather unusual property of this system is the apparent ability of one of these oscillators to drive activity at a previous phase position. This feature suggests a mechanism which retains a memory of phase displacement. A recently completed study provides additional evidence for such a mechanism (22). ACKNOWLEDGEMENTS This work was supported by Grant No. BNS-8601821 from the National Science Foundation. The author thanks Ms. Sharon Wittig for secretarial assistance, Mr. Richard Brunck for illustrations, and Mr. Charles Badland for photography.
PHASE SHIFTS O F F E E D I N G T I M E
995
REFERENCES 1. Amsel, A.; Work, M. S. The role of learned factors in "spontaneous" activity. J. Comp. Physiol. Psychol. 54:527-532; 1961. 2. Aschoff, J. Effects of periodic availability of food on circadian rhythms. In: Hiroshige, T; Honma, K. eds. Comparative aspects of circadian clocks. Sapporo: Hokkaido Univ. Press; 1987:19-40. 3. Aschoff, J.; yon Goetz, C.; Honma, K. Restricted feeding in rats: Effects of varying feeding cycles. Z. Tierpsychol. 63:91-111; 1983. 4. Boulos, Z.; Rosenwasser, A. M.; Terman, M. Feeding schedule and the circadian organization of behavior in the rat. Behav. Brain Res. 1:39-65; 1980. 5. Clarke, J. D.; Coleman, G. J. Persistent meal-associated rhythms in scn lesioned rats. Physiol. Behav. 36:105-113; 1986. 6. Coleman, G. J.; Harper, S.; Clarke, J. D.; Armstrong, S. Evidence for a separate meal-associated oscillator in the rat. Physiol. Behav. 29:107-115; 1982. 7. Krieger, D. T.; Hauser, H.; Krey, L. C. Suprachiasmatic nuclear lesions do not abolish food-shifted circadian adrenal and temperature rhythmicity. Science 197:398-399; 1977. 8. Ottenweller, J. E.; Tapp, W. N.; Natelson, B. J. Phase-shifting the light-dark cycle resets the food-entrainable circadian pacemaker. Am. J. Physiol. 258:R994-RI000; 1990. 9. Richter, C. P. A behavioristic study of the activity of the rat. Comp. Psychol. Monogr. 1:1-54; 1922. 10. Rosenwasser, A. M.; Adler, N. T. Structure and function in circadian timing systems: Evidence for multiple circadian oscillators. Neurosci. Biobehav. Rev. 10:431-448; 1986. l 1. Rosenwasser, A. M.; Pelchat, R. J.; Adler, N. T. Memory for feeding time: Possible dependence on coupled circadian oscillators. Physiol. Behav. 32:25-30; 1984. 12. Ruis, J. F.; Talamini, L. M.; Buys, J. P.; Rietveld, W. J. Effects of time of feeding on recovery of food-entrained rhythms during subsequent fasting in SCN-lesioned rats. Physiol. Behav. 46:857-866; 1989.
13. Satinoff, E.; Prosser, R. A. Suprachiasmatic nuclear lesions eliminate circadian rhythms of drinking and activity, but not of body temperature, in male rats. J. Biol. Rhythms 3:1-22; 1988. 14. Sheffield, F. D.; Campbell, B. A. The role of experience in the spontaneous activity of hungry rats. J. Comp. Physiol. Psychol. 47:97100; 1954. 15. Sokolove, P. G.; Bushell, W. N. The Chi Square Periodogram, its utility for analysis of circadian rhythms. J. Theor. Biol. 72:13 l - 160; 1978. 16. Stephan, F. K. Limits of entrainment to periodic feeding in rats with suprachiasmatic lesions. J. Comp. Physiol. [A] 143:401-410; 198 I. 17. Stephan, F. K. Circadian rhythm dissociation induced by periodic feeding in rats with suprachiasmatic lesions. Behav. Brain Res. 7: 81-98; 1983. 18. Stephan, F. K. Circadian rhythms in the rat: Constant darkness, entrainment to T cycles and to skeleton photoperiods. Physiol. Behav. 30:451-462; 1983. 19. Stephan, F. K. Phase shifts of circadian rhythms of activity entrained to food access. Physiol. Behav. 32:663-671; 1984. 20. Stephan, F. K. Coupling between feeding- and light-entrainable circadian pacemakers in the rat. Physiol. Behav. 38:537-546; 1986. 2 I. Stephan, F. K. Forced dissociation of activity entrained to T cycles of food access in rats with suprachiasmatic lesions. J. Biol. Rhythms 4:467-479; 1989. 22. Stephan, F. K. Resetting of a circadian clock by food pulses. Physiol. Behav. 52:997-1008; 1992. 23. Stephan, F. K.; Swann, J. M.; Sisk, C. L. Anticipation of 24 hr feeding schedules in rats with lesions of the suprachiasmatic nucleus. Behav. Neural Biol. 25:346-363; 1979. 24. Stephan, F. K.; Swann, J. M.; Sisk, C. L. Entrainment of cireadian rhythms by feeding schedules in rats with suprachiasmatic lesions. Behav. Neural. Biol. 25:545-554; 1979. 25. Winfree, A. T. Integrated view of resetting a circadian clock. J. Theor. Biol. 28:327-374; 1970.
0031-9384/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
Printedin the USA.
Resetting of a Feeding-Entrainable Circadian Clock in the Rat F R I E D R I C H K. S T E P H A N
Department of Psychology, Psychobiology-Neuroscience Program, Florida State University, Tallahassee, F L 32306-1051 Received 7 April 1992 STEPHAN, F. K. Resetting of a feeding-entrainable circadian clock in the rat. PHYSIOL BEHAV 52(5) 985-995, 1992.-Reentrainment of anticipatory activity (AA)after phase shifts of food access was studied in rats with lesionsof the suprachiasmatic nuclei. Eight- or 10-h phase delays of feeding time resulted in delaying transients of AA. Twelve-h phase shifts and some 10-h phase advances resulted in rapid reentrainment (2-3 days) without visible transients. Most phase advances of 10 h resulted in delaying transients while 8-h advances induced transients with advancing and delaying components in a number of rats. Split transients were not prevented by advancing mealtime in 1-h steps. Phase shifts of food in multiple steps failed to accelerate delay shifts but retarded advance shifts. After the first 8-h phase delay shift, increased activity reappeared at the preshift phase of AA, simultaneously with anticipation of the phase-shifted meal time. The observation of split transients indicates that two or more circadian oscillatorsmediate entrainmentto mealtime, and the reappearance of AA at a previously establishedphase suggest the possibilitythat this system has a memory of phase displacement. Circadian rhythms
Entrainment
Phaseshifts
Suprachiasmaticnuclei
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transients. Finally, a few rats show a combination of delaying transients and simultaneously AA to the mealtime (19). The purpose of the present study was to conduct a more extensive investigation of the resetting of AA in rats with SCN lesions. It should be noted that phase shifts of food access have been studied in intact rats, but the results are influenced by the presence of the light-entrainable circadian pacemaker which retards the reentrainment of AA (20). The emphasis of the present study was to reexamine resetting of AA to 8-h phase advances and delays. Ten-h and 12-h phase shifts were also imposed to cover a broader range of phase displacements. In addition, single step phase shifts were compared with multiple step phase shifts in order to test whether the rate and direction of transients could be controlled. Stepwise phase shifts correspond to a temporary T cycle (T = period of food access), and because the upper limit of entrainment is near 31 h (4,16), it seemed possible that large phase delays could be induced to accelerate reentrainment and that multiple step advances might prevent delaying transients. Finally, the phase stability of AA following reentrainment was assessed by imposing 1-3 days of food deprivation immediately after restricted feeding or after ad lib feeding.
THE ability of rats to anticipate a single meal per day by an increase in activity beginning several hours prior to mealtime was reported almost 70 years ago (9). During the past two decades it has become increasingly clear that this anticipatory activity (AA) reflects the entrainment of a circadian pacemaker that is anatomically and functionally distinct from the circadian pacemaker that entrains to light-dark cycles. First, lesions of the suprachiasmatic nucleus (SCN) which abolish many (but perhaps not all) circadian rhythms in rats maintained in ad lib feeding conditions [for review, see (10)] do not abolish the rise in temperature or serum corticosterone (7) or the development of AA prior to mealtime (4,16,23). Second, the limits of entrainment to mealtime are in the circadian range (22 to 31 h) (4,16), and the upper limit of entrainment to mealtime is considerably longer than for light pulses (18). In addition, the onset of AA relative to food access depends systematically on the period of feeding, thus representing the phase angle of entrainment (2,3). Third, although AA rarely persists into ad lib feeding conditions, it nearly always appears during total food deprivation, even after many weeks of intervening ad lib conditions (5,6,8,11). Fourth, following phase shifts of food access, AA reentrains gradually over several cycles (19), and such transients are characteristic of endogenous oscillators. However, several aspects of reentrainment appear to be different from those observed after phase shifts of LD cycles. In particular, 6- or 8-h phase advances of mealtime induce three modes of reentrainment. Some rats display delaying transients, i.e., the system responds as if the advances were 18- or 16-h delays, respectively. Other rats anticipate the phase-shifted mealtime by the second or third day without clearly discernable
METHOD
Animals, Housing, and Activity Recording Sixteen male adult Sprague-Dawley rats (mean body weight 290 g; SEM + 6.8 g) with SCN lesions were housed individually in sound-attenuated chambers that enclosed an activity wheel with a small adjacent cage. An exhaust fan provided fresh air flow and masking noise. Water was available ad lib throughout the study and food access was controlled by a motorized gate.
985
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The chambers and the experimental room were maintained in DD. During cleaning (weekly) and restocking of food hoppers (at 3-5-day intervals at irregular times of day) the door of the experimental room was left ajar. The brief exposure to low levels of illumination from an adjacent room had no discernable systematic effect on activity. Wheel revolutions were recorded by means of an infrared light-emitting diode and a phototransistor mounted across a tab on the running wheel. Contact circuits recorded licks and eating. All three measures were monitored continuously by a computer and cumulative counts were stored on disk every 10 min. The motorized food gate was under computer control.
phase shift consisted of a 10-h phase advance in a single step for the first group and in ten 1-h steps for the second group. The first group was then food deprived for 3 days, followed by 8 days of ad lib feeding and 2 additional days of food deprivation (see Figs. 1 and 2). Because the rats in the second group required much longer to reentrain and only three rats produced useful data for the complete experiment, the last food deprivation procedure was not used for this group. RESULTS
Histology and Periodicity Analysis
Rats were anesthetized with sodium pentobarbital (5.2 mg/ 100 g b.wt.) and received 0.05 cc atropine sulfate (IM). Electrolytic lesions were aimed at the SCN. With the incisor bar 5 mm above the interaural line, stereotaxic coordinates were 1.3 mm anterior to bregma, _+0.2 mm lateral, and 9.6 mm ventral to the dura. A 2 mA anodal direct current was passed for 18 s through a stainless steel electrode (0.4 mm diameter) which was insulated except for 0.5 mm at the tip. A total of 26 rats received attempted SCN lesions. Drinking was monitored for 3-4 weeks in standard hanging cages, and 16 rats that had the most disrupted circadian rhythms were selected for the experiment.
Based on light microscopic examination, no SCN tissue was identified in any of the 16 rats. The lesions had been made deliberately large so that portions of the anterior hypothalamic area, medial preoptic area, retrochiasmatic area, and the optic chiasm sustained damage. The success of the lesions was also evaluated by examining the last 12 days of drinking prior to transferring the animals to the activity wheels and the first 8 days of ad lib conditions in the wheels with the chi-square periodogram. None of the animals had significant peaks between 18 and 30 h. Drinking data were used because some rats had very low levels of wheel running in baseline conditions which could have made it difficult to detect residual circadian rhythmicity.
Histology
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Rats were deeply anesthetized with sodium pentobarbital and perfused intracardially with normal saline, followed by 10% formalin. Brains were removed and stored in sucrose formalin for at least 10 days and then embedded in gelatin. Frozen sections were cut in the coronal plane at 40 um through the region of damage. Every fourth section was mounted on slides and stained with cresyl violet for light microscopic examination.
Eight-hour delays. All 8-h delays of food access resulted in delaying transients of anticipatory activity (see Figs. 1-4). (The data from rat M60-2 could not be used because of very low activity levels.) The number of days required to reentrain to the single-step delay was between 4 and 6 days on the first shifts. Rats which were exposed to this procedure on the second or third shift required on the average only 3 days to reentrain AA (range 2-5 days; Figs. 1-4). After the first phase delay, increased activity appeared near the preshift phase position of AA some 2-7 days after the day of the phase shift (Figs. 1 and 2). This persisting activity will be discussed separately below. For twostep delays, no consistent effect on days to reentrainment occurred (range 2-6 days, Figs. 1 and 2, 3rd delay), whereas fourstep delays obviously prevented reentrainment in fewer than 4 days (Fig. 1, 2nd delay; Fig. 2, 1st delay). The event records, as well as the activity pattern averaged over four 26-h days (Fig. 5, top), show that AA of five rats followed the daily 2-h delays. In two rats (M60-4, M60-5), the onset of AA shifted by less than 2 h per day so that the onset of AA preceded mealtime by about 8 h (Fig. 5, top). After the first phase shift, the phase angle of entrainment (onset of AA to mealtime) was reduced from about 5 h to 3 h following reentrainment. This effect is visible in the event records and in the distribution of activity averaged over 6 days preshift and days 11-17 postshift (Fig. 6, top row). The peak level of AA was also reduced in six of seven rats following reentrainment. In part, this effect is due to the absence of AA on some of the days used for averaging. Ten-hour delays. Ten-h delays resulted in delaying transients in all seven rats exposed to this procedure (one rat died prior to this shift). While the onset of AA delayed systematically in all rats, in three rats the activity was split into two bouts on the second day. The second bout began about 3 h prior to the new food access time (see Fig. 3, M61-1; Fig. 4, M61-8). On the average, reentrainment occurred in 4 days (range 3-5 days). Twelve-hour phase shift. Only one of seven rats displayed delaying transients to the 12-h phase shift (Fig. I, M60-4). In the remaining rats, AA persisted at the pre-shift phase for one
Surgery
Data Analysis Event records of wheel running, drinking, and contacts with the food hopper were double plotted (48 h horizontal time scale). Wheel running records were the primary measure in the experiment. For the illustrations, the vertical height was plotted as the square root of the number of revolutions per 10 min. Zero counts were printed as one pin and maximum symbol height was clipped at ->81 responses (9 pins). For selected data segments, the amount of activity was assessed by averaging 30-min values over a number of consecutive days. The effect of SCN lesions on circadian rhythms was assessed with the chi-square periodogram (15). PROCEDURE Animals were kept in ad lib feeding conditions for 8 days. To ease the transition to restricted feeding, food access was limited to 4 h/day (1400-1800) for 8 days. The access time was then reduced to 2 h (1400-1600) for 14 days to establish stable anticipatory activity. Eight rats were then exposed to three consecutive 8-h phase delays of food access, in a single step, in four 2-h steps, or in two 4-h steps (see Figs. 1 and 2). Eight rats were exposed to two consecutive 8-h phase advances, in one 8-h step or in eight 1-h steps. The third phase shift was an 8-h delay (see Figs. 3 and 4). The first group was then exposed to a 12-h phase shift and the second group to a 10-h delay of food access time. Eight days after these phase shifts, all rats were food deprived for 3 days, followed by 10 days ofad lib feeding and then placed on restricted feeding at the previous phase position. The last
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to three days and then vanished while AA to the phase shifted food access appeared in 2 to 4 days (Fig. 1, M60-3; Fig. 2; M605 and M60-7). Persisting activity. A surprising result was the appearance of a band of activity beginning near the preshift phase of AA after the initial 8-h phase delay. This activity emerged 2 to 7 days after the phase shift and sporadically disappeared and reappeared (Figs. 1 and 2). On most days, the activity began near the preshift phase of AA. As shown by the distribution of activity averaged over days 11-17 postshift, the amount of this secondary activity was substantial, and in some cases approached preshift levels (e.g., Fig. 6, top row, dashed lines).
Phase Advances Eight-hour advance. None of the rats exposed to one-step advances displayed daily advancing transients between the old and the new phase. AA to the phase shifted access time appeared within 2-3 days. At this point in most rats, the onset of AA continued to advance over the next 2-3 days (e.g., Fig. 3, M61-
6 shows the clearest indication of advancing transients after the second phase shift) and appeared reentrained between 3-5 days. For four rats exposed to the 8-h advance in l-h steps, AA advanced on a daily basis, indicating entrainment to a 23-h T cycle (e.g., Fig. 3, M61-4, 2nd advance). Four rats displayed AA on the first 2-3 and on the last 2-3 days of the shift but did not anticipate food access for 1-3 days in between (e.g., Fig. 3, M611, 2nd advance). In these rats the duration of AA was very short, indicating that the system was very close to the limits of entrainment. The distribution of activity averaged over 8 consecutive 23-h days clearly shows the presence of AA, although the average level of activity is low for rats which did not display AA every day (Fig. 5, bottom). Both types of phase shift were accompanied by a secondary component of activity in 15 of 16 instances. In five cases, this component delayed until it reached the new phase of food access (e.g., Fig. 3, M61-1, 2nd advance; M61-6, 1st advance; Fig. 4, M61-7, 1st and 2nd advance). Since these rats also displayed advancing transients or AA entrained to a 23-h T-cycle, this
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FIG. 6. Distribution of wheel running averaged over 6 days prior to the first phase shift (solid lines, open bars) and for days 11-17 after the first phase shift (interrupted lines, hatched bars). Three rats with high levelsof secondary activity (interrupted line near open bar) from the 8-h delay group (top) and the 8-h advance group (bottom) are shown.
mode of reentrainment involves splitting of transients. In rat M61-7 (Fig. 4), this activity trails the phase shifted food access and appears to delay a second time. In other rats, unstable bands of activity occurred after the phase shifted meal time (e.g., Fig. 3, M61-4, 1st advance). Ten-hour advance. In response to the 10-h advance in a single step, two of seven rats displayed systematic delaying transients (Fig. 1, M60-3, M60-4, last phase shift). Three rats showed a response similar to that observed after 12-h phase shifts. AA continued for 2 days at the preshift phase and then vanished. AA appeared at the new phase after 2-3 days without intervening transients (e.g., Fig. 2, M60-5). The other two rats showed a single delaying transient on the second day after the shift (e.g., Fig. 2, M60-7). Due to equipment problems, only four rats could be evaluated for the 10-h advance in 1-h steps. One of these entrained to the T = 23-h cycle. This rat was a replacement which had not been exposed to previous phase shifts. A second rat entrained for 6 days but then lost entrainment (Fig. 4, M61-8). One rat displayed delaying transients which continued through the T cycle and then failed to entrain once more after the new phase position of the meal had been established. Entrainment finally occurred when the transients approached meal access for the third time (Fig. 4, M61-7). For 3 days, delaying transients and AA to the phase shifted meal can be seen simultaneously. The fourth rat displayed bouts of activity 3-4 h after the mealtime on 7 of the 10 days of the T cycle (Fig. 3, M61-4).
Phase Stability Following reentrainment after the 12-h phase shift, seven rats were food deprived for 2 days. The onset of AA on the second day had shifted less than 1 h in five rats (e.g., Fig. 1, M60-3, M60-4) but appeared delayed by about 3 h in the other two rats (Fig. 2, M60-5; M60-7). The 10-h delay shift was also followed by 2 days of food deprivation. One rat (M61-1) appeared in poor condition and was sacrificed on the second day. Because food was returned after the onset of AA on the third day, phase stability could be assessed for 3 cycles. Two rats showed successive phase advances (Fig. 3, M61-4; Fig. 4, M61-8), two showed successive delays, and two retained a fairly stable phase position (e.g., Fig. 4, M61-7). Twenty-two days after the 10-h advance in a single step, seven rats were food deprived for 3 days, followed by 8 days of ad lib feeding and 2 additional days of food deprivation to again assess the phase stability of AA. During the first food deprivation segment, AA remained near the established phase, or at least within the variability of AA during restricted feeding in all rats (e.g., Figs. 1 and 2). During the second food deprivation segment, AA had shifted by as much as 8 h from the last entrained phase position (e.g., Figs. 1 and 2, last FD condition). Eating and drinking. The contact eatometers used were not completely reliable for all rats and do not provide quantitative information about food intake. Nevertheless, as far as could be determined, rats contacted the food hopper at all meals, regardless of the presence of absence of AA or the type of transients after phase shifts.
PHASE SHIFTS OF FEEDING TIME
993
Event records of drinking show that many rats displayed anticipatory licking in addition to anticipatory wheel running. Furthermore, the pattern of secondary activity components as well as split transients was also reflected in drinking. This indicates that these responses are not peculiar to wheel running but reflect a more general activation of behavior. Representative examples are shown in Fig. 7. Bodyweight. At perfusion, the mean body weight (N = 14) was 367 g, SEM _+ 14.3 g. All rats had gained weight during the 156 days of manipulation of food access. DISCUSSION The present results confirm and extend previous observations regarding reentrainment of AA in rats with SCN lesions (19).
Phase delays of feeding time by 8 or 10 h induce delaying transients and rapid reentrainment of AA. For single-step phase delays, reentrainment was faster on the second and third shift than on the first shift. While this effect could not be assessed statistically because of the small N, an earlier study dealing with the limits of entrainment suggests a strong history dependence of the feeding-entrainable pacemaker, i.e., the upper limit, was at least 31 h when T was changed sequentially from 24 to 27, 29, and 31 h, whereas no rat entrained AA when T was abruptly changed from 24 to 31 h (16). The strong tendency of this pacemaker to delay was also confirmed since 8- or 10-h phase advances induced delaying transients in some rats. Eight-h phase delays in two or four steps had little effect on the rate of reentrainment, except to prevent reentrainment in
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FIG. 7. Double-plotted segments of event records of drinking for two rats exposed to 8-h delays (top) or 8-h advances (bottom) of food access. Compare the close correspondence of the temporal pattern of drinking with that of wheel running shown in Figs. 1 and 3. Dashed lines indicate potential splitting of transients for M60-3.
994 fewer than 4 days when 2 h steps were used. These results suggest that this pacemaker rapidly slows down when mealtime is delayed. Consequently, reentrainment takes place in a few days and cannot be accelerated by presenting the Zeitgeber at intermediate phases. The rate of reentrainment to single step 8-h or 10-h delays is surprisingly rapid, especially after the first delay shift, requiring only 4 days or less in 15 of 16 instances. Unusual modes of reentrainment were again observed with 8-h phase advances (19) and also with 10-h advances and 12-h phase shifts. One mode involved reentrainment without apparent intervening transients, particularly during the 10-h advances and 12-h phase shifts. Here, activity continued at or near the preshift phase for 1 or 2 days in a fashion similar to that of rats placed in total food deprivation (4,24). By the second or third day, the phase-shifted meal time was preceded by AA. This could be the result of strong resetting of the pacemaker (25) or alternatively, entrainment of a second oscillator at the new phase position and decoupling of activity from the oscillator at the previous phase position. A two-oscillator interpretation is supported by reentrainment consisting of split transients, which were particularly common during 8-h phase advances. In these cases, delaying transients could be observed concomitantly with advancing transients or apparent entrainment to food access (23-h T cycle). Since one oscillator cannot generate two different periods simultaneously, it seems most parsimonious to assume that two oscillators become temporarily uncoupled when the phase shifted Zeitgeber falls on a critical phase. The possibility that the feeding entrainable pacemaker consists of more than one oscillator is also supported by the ability of rats to anticipate two meals with different periods (e.g., 24 h and 24.5 h) per circadian cycle (17,21). Although few animals in this study showed activity after food access, termed succeeding activity (3), in a few cases one component of split transients appeared to arise from succeeding activity (e.g., Fig. 4, M61-7, 1st phase shift). This raises the possibility that a second oscillator, entrained at a lagging phase, is responsible for succeeding activity. The observation of AA during food deprivation revealed a considerable instability of phase after apparent resetting of the pacemaker. Following the 12-h phase shift and reentrainment, AA during food deprivation was either unshifted or delayed. This group of rats had only experienced phase delays before. The other group had been exposed to two-phase advances and a 10-h delay prior to food deprivation, and several rats showed advances in AA during food deprivation. This suggests that the phase shift history might influence the phase stability of the system in the absence of periodic cues. During the last food deprivation segment, rats expressed bouts of activity which could not readily be extrapolated to the previous entrained phase of AA. Either the pacemaker free ran through ad lib feeding at periods considerably longer or shorter than 24 h or the bouts were triggered by a second oscillator at a different phase position. In all rats, the onset of this activity was between 2300 h (Fig. l, M60-4) and 0600 h (Fig. 2, M60-5, M60-7), so that it is highly unlikely that it was triggered by external cues. An unexpected finding was the reappearance of activity near the preshift phase of AA after the first phase delay shifts. One explanation might be that these rats responded to an exogenous laboratory cue, because it is known that food-deprived rats become more sensitive to external disturbances (l, 14). This interpretation was used to account for recent observations that AA during food deprivation probes appeared near the last entrained phase when rats with SCN lesions were fed in the early afternoon,
STEPHAN but less consistently so when rats were fed at night (12). However, the authors point out that not all their findings are consistent with the external cue hypothesis and provide alternative explanations. In the present study, two factors argue against this interpretation. First, this activity did not appear every day and was also expressed on weekends, when potential noise cues are drastically reduced. Second, the onset of this activity was variable within as well as between animals. The only noise audible to human observers in the experimental room was from activity wheels. Consequently, the external cue hypothesis would require that animals respond to unspecified cues while ignoring audible cues from other rats. On the other hand, systematic advances and delays in the onset of these activity bouts over several cycles may indicate an underlying periodic process (see Fig. 7). Consequently, a tentative interpretation is that this activity is mediated by a second circadian oscillator, i.e., the initial phase delay shifts also may induce splitting, as was the case with 8-h phase advances. If a second oscillator is involved in generating this persisting activity, it becomes necessary to consider a mechanism which would permit the apparent return to a previous phase position, i.e., after the first phase shift the onset of the persisting activity was on the average near that of AA prior to the phase shift. Since it seems likely that the phase shifted Zeitgeber alters the phase (and period) of both oscillators, either a memory of previous phase or more likely of the amount of phase displacement would seem to be required to allow an oscillator to return to a prior phase position. Such a mechanism would have adaptive significance in that ifa food source disappeared from a usual time of day, a memory of time when food was previously available would be advantageous. Phase memory has been postulated previously, based on observations with intact rats which had been entrained to restricted feeding, followed by ad lib feeding and constant light or by a phase shift of the LD cycle. During food deprivation probes, AA emerged at a constant phase relationship to the free-running or to the phase-shifted activity rhythm (8,11). However, these results could also be accounted for by coupling between the feeding- and light-entrainable pacemakers which could preserve their phase relationship [see (20)]. In the present study, the light entrainable pacemaker is absent and, consequently, cannot serve as a phase reference. Since not all circadian rhythms may be abolished by SCN lesions (13), one possibility is that another circadian oscillator which is unaffected by periodic feeding could serve as an intemal phase reference. However, this study provides no direct information on this hypothesis. In summary, the results of the present study are consistent with the hypothesis that the feeding entrainable pacemaker (like the light-entrainable pacemaker) consists of more than one oscillator. The most compelling evidence for this interpretation is the occurrence of split transients, especially after 8-h phase advances, and possibly after 8-h delays of the Zeitgeber. A rather unusual property of this system is the apparent ability of one of these oscillators to drive activity at a previous phase position. This feature suggests a mechanism which retains a memory of phase displacement. A recently completed study provides additional evidence for such a mechanism (22). ACKNOWLEDGEMENTS This work was supported by Grant No. BNS-8601821 from the National Science Foundation. The author thanks Ms. Sharon Wittig for secretarial assistance, Mr. Richard Brunck for illustrations, and Mr. Charles Badland for photography.
PHASE SHIFTS O F F E E D I N G T I M E
995
REFERENCES 1. Amsel, A.; Work, M. S. The role of learned factors in "spontaneous" activity. J. Comp. Physiol. Psychol. 54:527-532; 1961. 2. Aschoff, J. Effects of periodic availability of food on circadian rhythms. In: Hiroshige, T; Honma, K. eds. Comparative aspects of circadian clocks. Sapporo: Hokkaido Univ. Press; 1987:19-40. 3. Aschoff, J.; yon Goetz, C.; Honma, K. Restricted feeding in rats: Effects of varying feeding cycles. Z. Tierpsychol. 63:91-111; 1983. 4. Boulos, Z.; Rosenwasser, A. M.; Terman, M. Feeding schedule and the circadian organization of behavior in the rat. Behav. Brain Res. 1:39-65; 1980. 5. Clarke, J. D.; Coleman, G. J. Persistent meal-associated rhythms in scn lesioned rats. Physiol. Behav. 36:105-113; 1986. 6. Coleman, G. J.; Harper, S.; Clarke, J. D.; Armstrong, S. Evidence for a separate meal-associated oscillator in the rat. Physiol. Behav. 29:107-115; 1982. 7. Krieger, D. T.; Hauser, H.; Krey, L. C. Suprachiasmatic nuclear lesions do not abolish food-shifted circadian adrenal and temperature rhythmicity. Science 197:398-399; 1977. 8. Ottenweller, J. E.; Tapp, W. N.; Natelson, B. J. Phase-shifting the light-dark cycle resets the food-entrainable circadian pacemaker. Am. J. Physiol. 258:R994-RI000; 1990. 9. Richter, C. P. A behavioristic study of the activity of the rat. Comp. Psychol. Monogr. 1:1-54; 1922. 10. Rosenwasser, A. M.; Adler, N. T. Structure and function in circadian timing systems: Evidence for multiple circadian oscillators. Neurosci. Biobehav. Rev. 10:431-448; 1986. l 1. Rosenwasser, A. M.; Pelchat, R. J.; Adler, N. T. Memory for feeding time: Possible dependence on coupled circadian oscillators. Physiol. Behav. 32:25-30; 1984. 12. Ruis, J. F.; Talamini, L. M.; Buys, J. P.; Rietveld, W. J. Effects of time of feeding on recovery of food-entrained rhythms during subsequent fasting in SCN-lesioned rats. Physiol. Behav. 46:857-866; 1989.
13. Satinoff, E.; Prosser, R. A. Suprachiasmatic nuclear lesions eliminate circadian rhythms of drinking and activity, but not of body temperature, in male rats. J. Biol. Rhythms 3:1-22; 1988. 14. Sheffield, F. D.; Campbell, B. A. The role of experience in the spontaneous activity of hungry rats. J. Comp. Physiol. Psychol. 47:97100; 1954. 15. Sokolove, P. G.; Bushell, W. N. The Chi Square Periodogram, its utility for analysis of circadian rhythms. J. Theor. Biol. 72:13 l - 160; 1978. 16. Stephan, F. K. Limits of entrainment to periodic feeding in rats with suprachiasmatic lesions. J. Comp. Physiol. [A] 143:401-410; 198 I. 17. Stephan, F. K. Circadian rhythm dissociation induced by periodic feeding in rats with suprachiasmatic lesions. Behav. Brain Res. 7: 81-98; 1983. 18. Stephan, F. K. Circadian rhythms in the rat: Constant darkness, entrainment to T cycles and to skeleton photoperiods. Physiol. Behav. 30:451-462; 1983. 19. Stephan, F. K. Phase shifts of circadian rhythms of activity entrained to food access. Physiol. Behav. 32:663-671; 1984. 20. Stephan, F. K. Coupling between feeding- and light-entrainable circadian pacemakers in the rat. Physiol. Behav. 38:537-546; 1986. 2 I. Stephan, F. K. Forced dissociation of activity entrained to T cycles of food access in rats with suprachiasmatic lesions. J. Biol. Rhythms 4:467-479; 1989. 22. Stephan, F. K. Resetting of a circadian clock by food pulses. Physiol. Behav. 52:997-1008; 1992. 23. Stephan, F. K.; Swann, J. M.; Sisk, C. L. Anticipation of 24 hr feeding schedules in rats with lesions of the suprachiasmatic nucleus. Behav. Neural Biol. 25:346-363; 1979. 24. Stephan, F. K.; Swann, J. M.; Sisk, C. L. Entrainment of cireadian rhythms by feeding schedules in rats with suprachiasmatic lesions. Behav. Neural. Biol. 25:545-554; 1979. 25. Winfree, A. T. Integrated view of resetting a circadian clock. J. Theor. Biol. 28:327-374; 1970.
