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Resetting of a circadian clock by food pulses
Physiology&Behavior,Vol. 52, pp. 997-1008, 1992
0031-9384/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
Printedin the USA.
Resetting of a Circadian Clock by Food Pulses 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 circadian clock byfood pulses. PHYSIOL BEHAV 52(5) 997-1008, 1992.--Rats with lesions of the suprachiasmatic nuclei were exposed to daily feeding until anticipatory activity (AA) developed. Meals were then phase advanced or delayed and presented for 1--4consecutive days. The phase of the circadian pacemaker was assessed during probes of total food deprivation before or after 8 days of intervening ad lib feeding. One or two food pulses caused phase delays for one cycle but were insufficientto reset the feedingentrainable pacemaker. Complete or partial resettingto 6- or 9-h advances or delays was observed in some rats after three food pulses and in all rats after four food pulses. In some rats, phase shifts of meals appeared to induce both advancing and delaying transients, and two bouts of AA appeared during food-deprivation probes. This suggests that the feeding entrainable pacemaker consists of two or more oscillators which became uncoupled after phase shifts. The persistence of AA at the preshift phase observed after initial phase delays, concomitantly with AA to the phase shifted meals, also suggests the presence of a second oscillator. Circadian rhythms
Suprachiasmaticnuclei
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RATS maintained on a single meal per day display an increase in activity beginning several hours prior to mealtime (9). This anticipatory activity (AA) persists in rats with lesions of the suprachiasmatic nuclei (SCN) which abolish many circadian rhythms in ad lib-fed rats, and considerable evidence indicates that AA is mediated by a circadian pacemaker that is anatomically distinct from the light-entrainable circadian pacemaker (presumably the SCN) [for review, see (10)]. The formal circadian properties of the feeding-entrainable pacemaker are best studied in rats with SCN lesions because the two circadian systems interact, albeit only weakly (18-20). Several important circadian properties of AA have been established in rats with SCN lesions. First, AA develops only when the period of meals is between 22 and 31 h (2,15,24). The upper limit of entrainment is greater if the period is increased in several steps, implying history dependence of the pacemaker (15). Second, the onset of AA relative to mealtime systematically increases with period (1), suggesting that this measure represents the phase angle of entrainment. Third, although AA rarely persists (i.e., free runs) in ad lib feeding conditions, it persists for up to 5 days during food deprivation (2). Furthermore, AA reappears during food deprivation even after weeks of intervening ad lib feeding at a phase near that of preceding entrainment (4,5,7,11). This suggests that the pacemaker free runs but that activity becomes decoupled in ad lib feeding. Fourth, following phase shifts of mealtime, several days are required until AA reentrains to the new mealtime (17,23). Transients between two steady states of entrainment are characteristic of oscillators, and can provide considerable insight into the dynamics of circadian clocks. A formal description of the effects ofa Zeitgeber on circadian oscillators can be obtained by establishing a phase-response curve (PRC) which displays the phase displacement resulting from a Zeitgeber delivered only
997
Food pulses
once at various circadian times. This technique has long been used to study light-entrainable circadian rhythms (8) and has also been used to assess the effects of dark pulses (3), as well as nonphotic signals [e.g., (6)]. As yet, no PRC for meals has been established. One of the major reasons is that AA fails to free run in ad lib feeding conditions and a state of food deprivation cannot be imposed for a sufficient number of days to use a standard protocol which assesses the effect of a single presentation of the Zeitgeber on the free-running rhythm. The present study was an attempt to study resetting behavior by a method which, while not equivalent to a standard PRC, would nevertheless provide some insight into the resetting dynamics of the feeding-entrainable circadian pacemaker. The method selected for the first experiment was to assess the phase displacement resulting from exposure to one phaseshifted signal after steady-state entrainment. Since AA is expressed during total food deprivation, the phase-shifted signal was followed by 3 days of food deprivation. Because the phase of AA might not yet have attained a steady state in 3 days, this was followed by 1 week of ad lib feeding and 3 more days of food deprivation [c.f., (4)]. The results of the first experiment indicated that a single food pulse was insufficient to reset the circadian pacemaker. Consequently, in a second experiment, the phase displacement in response to two, three, or four consecutive food pulses was assessed. METHOD
Animals, Housing, and Data Collection Twenty male Sprague-Dawley rats (body weight approximately 300 g) with SCN lesions were housed in individual soundattenuated chambers that enclosed an activity wheel with a small adjacent cage. An exhaust fan provided fresh air flow and mask-
998 ing noise. The chambers and the experimental room were maintained in constant darkness. Water was available ad lib throughout the study and bottles were refilled at 2- to 3-day intervals without opening the chambers. Food hoppers were filled with pellets (Purina Laboratory Rat Chow, No. 5001) at 3- to 5-day intervals at irregular times between 0900 and 1700 h. During replacement of bedding (weekly) and restocking of food hoppers, the door of the experimental room was left ajar. Although the brief exposure (<1 min) to low levels of light has little or no effect on the activity of rats with SCN lesions, the maintenance procedures did induce brief episodes of wheel running in some rats (see the Results and Discussion section). Wheel revolutions were recorded by means of an infrared diode and a phototransistor mounted across a tab on the wheel. Licks were recorded with contact circuits. Sixteen chambers were also equipped with contact circuits on the food hopper grid. All measures were monitored continuously by computer, and counts were stored on disk every l0 min. Food access was controlled by a motorized gate that was activated by computer.
Surgery 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. The incisor bar was 5 mm above the interaural line, and the stereotaxic coordinates were 1.3 mm anterior to bregma, +0.2 mm lateral, and 9.6 mm ventral to dura. A 1.5 mA anodal direct current was passed for 15 s through a stainless steel electrode (0.4 mm diameter) that was insulated except for 0.5 mm at the tip. A total of 24 rats received attempted SCN lesions. Drinking was monitored for 2-3 weeks in standard hanging cages, and 20 rats with the most disrupted circadian rhythms were selected for the two experiments.
Histology 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 tsm through the region of damage. Every fourth section was mounted on slides and stained with cresyl violet for light microscopic examination.
Data Analysis Event records of wheel running, drinking, and contacts with the food hopper were double plotted (48-h horizontal time scale) on a dot matrix printer. 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 to 3 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 counts over a number of consecutive days. The expression of AA during food deprivation probes was determined from event records and printouts of 10-min data counts. A bout of AA was defined by sustained activity lasting 4-8 h with pauses less than 1 h, representing a tenfold increase over activity in the preceding and succeeding 4 h. In a few cases, bouts falling short of these criteria were accepted on the basis of consistency across rats in the same group. The effect of SCN lesions on circadian rhythms was assessed with the chi-square periodogram (14).
STEPHAN PROCEDURE
Experiment 1 Four rats were placed in the activity wheels on ad lib food for 10 days. One rat died during this phase of the experiment. For the remaining three rats, food access was then limited to 2 h/day (1200-1400 h). After stable entrainment of AA, food was presented with a 4-h delay. This was followed by 3 days of food deprivation, 8 days of ad lib feeding, and 3 additional days of food deprivation. Two h/day feeding was then resumed at the new phase. When AA appeared stable, phase shifts were repeated using delays of 6, 8, and l0 h. Three, or 8 additional days ofad lib feeding, were added prior to the resumption of restricted feeding to facilitate recovery from food deprivation (see Fig. l ). A 1-h phase advance was inadvertently imposed when the computer clock was reset to standard time.
Experiment 2 Sixteen rats were placed in the activity wheels with ad lib food for 20 days. Food access was then reduced to 3 h/day for 29 days. Four rats each were then exposed to phase delays or to phase advances of mealtime of 6 or 9 h. To reduce the potential effects of noise cues from running wheels among rats, two rats exposed to phase delays were housed alternately with two rats exposed to phase advances. First, the phase shifted meal was presented for 2 consecutive days. This was followed by 8 days of ad lib feeding and 3 days of food deprivation. Restricted feeding was then resumed at the new phase position. This procedure was repeated except that the phase shifted meals were presented for three and then for four consecutive meals. During the last phase of the experiment, rats were exposed three or four times to a single day of food deprivation interspersed with ad lib feeding (see Figs. 2, 4-6). RESULTS AND DISCUSSION
Histology and Periodicity Analysis Light microscopic evaluation of the lesions indicated complete destruction oftbe SCN in the three rats used in Experiment 1. Periodicity analysis of drinking over 9 days of ad lib feeding showed no significant peaks between 4 and 30 h. Of the 16 rats used in Experiment 2, 15 brains were available for light microscopic examination. The SCN appeared totally destroyed in 13 rats. In these rats, periodicity analysis of drinking over 10 days of ad lib feeding showed no significant peaks in the circadian range, although one rat (#60-4) had uitradian peaks at 8 and 12 h and another (#60-8) at 15 h. In two rats (#61-3, #61-7) a caudal unilateral SCN remnant remained intact. Both rats had low peaks at 25 h in the periodogram. Drinking data were used for periodicity analysis because low levels of activity in some rats would have made it difficult to detect residual rhythms. The absence of free-running activity rhythms is readily apparent in the event records (Figs. 1, 2, 4-6).
Experiment 1: Effects of Single Food Pulses Several difficulties in interpreting the results must be noted at the outset. First, since the phase shifts of the Zeitgeber follow an entrained condition, the pacemaker is presumably not at its endogenous period and it is difficult to predict a priori what its period will be following a phase perturbation, i.e., the pacemaker could simply start to free run, or, because of history dependence, return to a period close to 24 h. Second, since AA rarely persists in ad lib conditions (see the Introduction section), the actual trajectory of the pacemaker to the phase of AA during food
RESETTING BY FOOD PULSES
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show the beginning and end of ad lib feeding. Phase shifts in hours are given in the left margin. AL = ad lib food, FD = food deprivation. The vertical line marks the initial phase of AA and interrupted lines mark the estimated phase during ad lib feeding.
deprivation probes must be inferred in most cases. Over 8 days ofad lib feeding, ambiguities may arise because, for example, a period of 23.5 h would lead to the same phase as a period of 26.5 h. Third, although there is considerable evidence that the pacemaker consists of two (or more) oscillators ( 16-19,23), few rats express two separate bouts of AA during food deprivation after forced dissociation (16,22). Consequently, presumed trajectories of a second oscillator are highly subjective. Finally, it is not clear whether transients indicate the phase of the pacemaker or of a driven process, i.e., the pacemaker may be fully reset on the first cycle while the driven process may require a number of cycles to catch up, generating transients. Because the phase of the pacemaker cannot be assessed directly, the phase of AA was taken to coincide with that of the pacemaker or one of its oscillators, although this may not actually be the case. A double-plotted event record for one of the three rats in Experiment 1 is shown in Fig. 1. Prior to the first phase shift, all rats had developed AA and the onset of AA was between 0600 and 0800 h. The effect of the phase-shifted food pulse was assessed to the nearest 0.5 h by comparing the onset of AA on the day of the shift with that on the subsequent day. The onset of AA was delayed by 1.5 h in two rats and by 0.5 h in one rat (Table 1). During the subsequent 2 days of food deprivation, the onset of AA occurred earlier and preceded the preshift phase of AA by about 2 h (Fig. 1). During the second food deprivation probe, distinct bouts of activity began between 0600 and 0800 h, slightly later than on the last day of the previous food deprivation probe. This suggests that an oscillator free ran with a period >24 h during ad lib feeding. The onset of AA clearly shows that the phase of the pacemaker had not been delayed in response to the phase shifted food pulse. When restricted feeding was resumed at the new phase position, activity initially increased between 0800 and 0900 h, i.e., near the phase of AA at the preceding food deprivation probe. All three rats continued to show this activity on some days but not on others. Since maintenance was performed during the day and some rats ran in wheels while food hoppers were restocked, this induced activity contributed to the increase in activity on some days. However, maintenance-induced activity typically lasted only 10 to 60 rain, rather than 6 to 8 h. The potential influence of maintenance will be more fully discussed in Experiment 2. The presence of the early onset of AA prevented a clear assessment of the effects of the 6-h delay of mealtime. On the day after the phase shift, AA consisted of multiple dispersed bouts of activity without a distinct onset. On days 2 and 3 of food deprivation, distinct bouts of AA appeared between 0600 and 0730 h, a time comparable to that during the preceding food deprivation probe. During the second food deprivation probe after ad lib feeding, AA had advanced even more in all three rats, suggesting an oscillator period of <24 h (Fig. l, second FD after the 6-h delay). When restricted feeding was resumed, AA appeared to reentrain by delaying transients, the origin of which could be projected back to the phase of AA during food deprivation (Fig. 1). AA was absent for the initial 2 to 3 days of RF, and none of the rats achieved stable entrainment the first time the apparent transients approached mealtime. Rat M51-3 (Fig. 1) seems to show delaying transients a second time before stable AA was established. During the last 10-15 days on RF, all three rats showed fairly stable AA beginning 4-5 h prior to mealtime, as well as bouts of lower levels of activity beginning between 0700 and 1000 h. While some of these early activity bouts could have been induced by maintenance, all three rats showed early onset AA on most weekend days as well (see Experiment 2). It should be
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RESETTING BY FOOD PULSES
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noted that it is highly unusual for rats with SCN lesions to require 15 days to entrain to mealtime. It seems likely that the prior phase shifts and the phase of AA during the preceding food deprivation probe was responsible for this effect. The effects of the 8-h delay pulse were also difficult to evaluate. In two rats, the major bouts of activity during food deprivation began near the phase-shifted food pulse (Fig. 1). However, since this phase was similar to that observed during the preceding food-deprivation probes, it is not likely indicative of resetting caused by the food pulse. One rat showed small dispersed bouts of activity without a clear onset. During the second food-deprivation probe after ad lib feeding, AA began between 0400 and 0600 h in the three rats, suggesting a period close to 24 h during ad lib feeding. After 8 additional days of ad lib feeding, restricted feeding was resumed and AA developed within 2-4 days. Rat M51-3 displayed an increase in activity after food access which was clearly influenced by the 1-h phase advance (Fig. 1). The effects of the 10-h delay were also difficult to ascertain. During the second and third day of food deprivation, the onset of AA occurred between 0300 and 0400 h in two rats and at 0700 h in rat M51-3 (Fig. l). During the second food-deprivation probe after ad lib feeding, the onset of AA was slightly delayed for one rat and virtually unchanged for the other two rats, indicating that the period of the pacemaker had remained near 24 h during ad lib feeding (Fig. 1). DISCUSSION These results indicate that exposure to a single-phase shifted food pulse can delay the phase of a feeding entrainable oscillator for 1 cycle (4-h delay shift), but is apparently insufficient to achieve a stable resetting of phase. The persisting activity near the initial phase of AA prevented a clear assessment of the effects of subsequent phase shifts. Apparent resetting of phase (e.g., Fig. l, 8-h delay) was most likely due to coincidence with the phase of AA during the earlier food deprivation probes. The experiment produced some unexpected results. At the second and third phase position of food access, an increase in activity occurred near the initial entrained phase of AA. While some of this activity could be attributed to maintenance, its appearance on weekends and transient like changes in its onset suggest that an endogenous oscillation may underly this activity. Furthermore, the onset of AA during the food deprivation probes was between 0300 and 0700 h, i.e., times when external disturbances are unlikely. Throughout the experiment, AA during food deprivation appeared near the initial phase of AA (Fig. 1, vertical line). During the 8 days ofad lib feeding, the phase of AA appeared relatively stable, generally advancing or delaying by less than 3
h. This indicates that the period of the oscillator was between 23.7 and 24.3 h during the 8 days ofad lib feeding. The major purpose of the second experiment was to investigate how many food pulses are required to reset the pacemaker. Because of the possibility that the unexpected results (i.e., apparent return to the preshift phase of AA) might have been influenced by the short food access time and by the food deprivation immediately after the phase shift, two changes in procedures were made. First, food access time was increased to 3 h, and the first food deprivation probe was omitted, i.e., rats were exposed to phase-shifted food pulses for 2, 3, or 4 days, with the last pulse marking the beginning of ad lib feeding for 8 days and then 2.5 or 3.5 days of food deprivation were imposed to assess the phase of AA. Food deprivation always began 12 h away from the phase-shifted mealtime. RESULTS
Phase Delay l--Two Pulses Representative event records for two rats each in the 6-h and 9-h delay condition are shown in Figs. 2 and 4. All rats displayed AA prior to the first phase shift. The effects of the phase-shifted food pulse were assessed by measuring (to the nearest 0.5 h) the onset of AA on the day of the shift and on the subsequent day (since the animals had not yet been exposed to the second pulse, the effect on the phase of the oscillator is like that in Experiment 1). The onset of AA was delayed by 1.5 h for all four rats exposed to the 6-h delay and by l to 2 h for rats exposed to the 9-h delay (Table l). After 8 days of ad lib feeding, the phase of AA was assessed during 3 days of food deprivation. In the 6-h delay group, the phase of AA on the first day of deprivation was close to that of the preshift phase of AA. On the next day, short bouts of activity occurred 6 to 8 h earlier and then delayed over the next 2 days (Fig. 2, FD-2). Rat #61-1 showed increased levels of activity during ad lib feeding, with a period close to 24 h which suggests the trajectory of an oscillator to the phase of AA on the first day of food deprivation. However, it seems rather unlikely that an oscillator would then advance by as much as 8 h in a single cycle, only to delay over the subsequent two cycles. The examination of drinking records showed an increased tendency to lick at a period of about 26 h. Consequently, the expression of AA during food deprivation could have resulted from splitting of two oscillators, one of which remained near the phase (and period) of the preshift phase of AA, while the other delayed. This explanation would also account for the onset of AA following resumption of restricted feeding at the new phase position, i.e., daily delays give the appearance of delaying transients (Fig. 2). During the next 26 days on restricted feeding, two of four rats in the 6-h delay group showed increased levels of activity
1002
STEPHAN
near the preshift phase of AA. As was the case in Experiment 1, this activity occurred during daytime hours (0800-1800 h) and could have been influenced by external cues. However, when the average distribution of activity on Saturdays and Sundays over 3 consecutive weekends was compared with that of the 2 preceding work days, the early onset of activity was present or absent in both (Fig. 3). In the 9-h delay group, two of the rats displayed a short bout of activity (at 0200 or 0400 h) on the first day of food deprivation (Fig. 4, #60-5, FD-1) which could have resulted from delaying transients (c.f., Fig. 2). In the third rat (Fig. 4, #61-6, FD-1), the bout appeared near the preshift phase, and in the fourth rat, activity was too dispersed to determine the onset of AA. During the next 3 days, the onset of AA for all four rats was between 0600 and 0800 h, near or earlier than the preshift phase of AA. Upon resumption of restricted feeding, a mechanical food gate failure rendered the data of rat #61-5 useless for 10 days. The other three rats showed elevated activity near the preshift phase and only low levels of AA to food access for 10-20 days (Fig. 4). As in the 6-h delay group, some of this persisting activity
could have been associated with maintenance, but it also occurred on weekends. AA to food access became more pronounced in the latter stages at this phase position, and at least in rat #61-6 (Fig. 4), this coincided with apparent delaying transients from the persisting AA. Clearly, two food pulses were not sufficient to reset the phase of AA after 6- or 9-h delays.
Phase Delay 2--Three Pulses Three food pulses were sufficient to elicit delaying transients in all four rats in the 6-h delay group (Fig. 2). During the food deprivation probe, the onset of AA varied widely among the four rats (Fig. 2, FD-2). For rat #61-1, AA began near the phase shifted food access time, indicating a possible resetting of one oscillator. However, in the other three rats, the onset of AA seemed unrelated to either the new or to the preshift phase of AA. Rather, AA appeared near the time of the previous food deprivation probe (e.g., Fig. 2, #60-2, FD-1, and FD-2). When restricted feeding resumed, three rats reestablished AA by the 2nd day. Rat #61-1 (Fig. 2) showed splitting of AA with 2 days of advancing and 3 days of delaying transients.
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FIG. 3. Daily distribution of wheel running for the four rats in the 6-h phase delay group at the second phase position of restricted feeding (food access from 1800-2100 h). The solid line shows wheel running averaged over 3 Thursdays and 3 Fridays, while the dashed lines shows activity averaged over 3 Saturdays and 3 Sundays. Note that the early onset of anticipatory activity for rats #60-2 and #61-1 occurs on workdays as well as on weekends.
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1004 In the 9-h delay group, one rat showed delaying transients, but two rats displayed AA to the second and third pulse without intervening transients (e.g., Fig. 4, #61-6). Low levels of activity prevented a clear assessment of transients in the other rat (Fig. 4, 60-5). The phase of AA during the food deprivation probe was near the new phase position in all four rats, indicating a possible reset and a free running period only slightly longer than 24 h during ad lib feeding (Fig. 4, FD-2). AA was quickly reestablished when restricted feeding was resumed. These results indicate that three food pulses were sufficient to induce a change in phase of AA large enough to qualify as resetting in five of eight rats after 6or 9-h phase delays.
Phase Delay 3--Four Pulses Prior to the third phase shift, all rats displayed stable AA and did not show any obvious residual effects of previous phase shifts. Most of the short bouts of activity between 0900 and 1600 h were related to maintenance. All rats in both groups showed daily delays to the four food pulses, and in seven of eight rats the onset of AA on day 4 was close to that expected for reentrainment (Figs. 2 and 4). In one rat, AA became dispersed into multiple bouts over some 14 h preceding food access (not shown). During food deprivation, the phase of AA in the 6-h delay group was near the new phase position (Fig. 2, FD-3) indicating that the phase of at least one oscillator had been reset by the four food pulses and that its period had remained close to 24 h during ad lib feeding. Rat #6 l- l (Fig. 3) showed enhanced activity during ad lib feeding, supporting this interpretation. AA was quickly reestablished when restricted feeding resumed, presumably because the phase of one oscillator was already near that of the new food access time. This indicates that in order to achieve a 6-h delay, four food pulses are sufficient to reset the phase of the system. In the 9-h delay group, rat M60-6 showed only dispersed activity. In two rats, the phase of AA was near the new phase on the first day but was advanced by some 6 h on the next 2 days, i.e., near the preshift phase of AA (Fig. 4, #61-6, FD-3). This may indicate splitting, because it is unlikely that a single oscillator would phase advance 6 h in one cycle. The fourth rat had very low activity counts due to a rubbing activity wheel (Fig. 4, #60-5). All four rats reestablished AA within a few days.
STEPHAN
Phase Advance 1--Two Pulses All rats displayed AA prior to the first phase shift. The effect of advancing mealtime on the phase of AA was assessed by comparing the onset of AA on the day prior to the phase shift with that on the day of the phase shift (because the Zeitgeber occurred prior to the entrained AA). All rats in both groups except #618 (Fig. 6) showed a delayed onset of AA on the day of the shift (Table 1), and none of the rats anticipated the second food pulse. During 3 days of food deprivation, the onset of activity was advanced compared to the preshift phase of AA in all rats (Figs. 5 and 6, FD-l). Because similar advances were observed in the phase delay groups, this response may not be due to partial resetting. After the resumption of restricted feeding, all but one rat (#61-3, Fig. 5) displayed AA within 3 days. Some rats showed brief bursts of activity after food access every 3-5 days which were associated with maintenance in most cases (e.g., Fig. 6, #61-8).
Phase Advance 2--Three Pulses The data for rat #61-4 in the 6-h phase advance group could not be used for the remainder of the experiment due to equipment failure. In the remaining rats, the phase advance again induced delays in the onset of AA on the day of the shift (Figs. 5 and 6). The magnitude of the delay was generally comparable to that induced by the first phase shift. On the next day, four rats showed further delays in AA (e.g., Fig. 6, #60-8). The three rats in the 6-h advance group showed AA to the third phase shifted pulse (Fig. 5), but no AA was observed in the 9-h advance group. During food deprivation, a complex pattern of AA was observed. In all rats, two bouts of AA occurred, in some cases on the same day (e.g., Fig. 6, #61-8, FD-2), or at very different phase positions on day 1 and clay 2 of deprivation (Fig. 5, #604; Fig. 6, #60-8, FD-2). The two bouts suggests splitting of two oscillators, and this interpretation is reinforced by apparent split transients upon resumption of restricted feeding in rat #60-8 (Fig. 6). AA was quickly reestablished in all seven rats when restricted feeding was resumed, but appeared much less stable in the 9-h advance group than in the 6-h advance group.
Phase Advance--Four Pulses Food Deprivation Probes Without Phase Shifts Due to a programming error, rats in the 6-h delay group were not exposed to food deprivation on the second day after restricted feeding was ended. During the 3 single days of food deprivation alternating with ad lib feeding, three rats showed AA at a phase close to the previous entrained phase of AA (Fig. 2, #60-2, #6 ll, FD-5, and FD-6). This seems to indicate that in the absence of phase shifts the period of at least one oscillator remains close to 24 h during ad lib feeding so that the phase changes observed at the preceding two food deprivation probes were due to resetting. However, in three of the four rats, a second bout of activity occurred at a different phase position. In rat #6 l-1 (Fig. 2), increased activity during ad lib feeding appears to converge on this second bout. The different phase positions of the two bouts could be accounted for by splitting. In the 9-h delay group, activity bouts during the four food deprivation probes appeared near or slightly in advance of the last entrained phase of AA in all four rats, indicating oscillator periods near 24 h (Fig. 4, FD-4, to FD-7).
On the day of the phase shift all rats showed a delayed onset of AA. The three rats in the 6-h advance group displayed AA to the second and subsequent food pulses with daily advancing transients (Fig. 5). Rats in the 9-h advance group anticipated the third or fourth pulse (Fig. 6). This indicates that the phase oftbe pacemaker, or at least one of its oscillators, had been reset in two to three cycles in the 6-h advance group and in three to four cycles in the 9-h advance group. As in previous studies (17,23), the advancing transients in AA seem to be masked by food access (i.e., AA is expressed only when its onset precedes food access). During ad lib feeding, rat #60-8 again showed enhanced levels of activity with a period of about 24.4 h, suggesting a possible trajectory of an oscillator (Fig. 6). On the first day of food deprivation, the phase of AA, compared to the day of the last food pulse, was delayed by 0.5 to 4 h, suggesting that the period of at least one oscillator remained near 24 h in all seven rats. The phase of AA on the second day remained fairly stable in most rats, but several rats showed a marked advance on subsequent days (e.g., Fig. 5, #60-4, FD-3), suggesting instability or splitting
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RESETTING BY FOOD PULSES despite the apparent resetting. The data for one rat (#60-3) became unusable at this stage because of mechanical problems with the activity wheel. AA was reestablished quickly when R F was resumed. Rat #60-4 showed delaying transients from the advanced bout of AA (Fig. 6). All remaining rats consistently anticipated food access until RF was terminated.
Food Deprivation Probes Without Phase Shifts During the first food deprivation probe, the onset of AA was at or slightly earlier than on the last day of restricted feeding in all six rats (Figs. 5 and 6, FD-4). However, during the three lday deprivation probes following 10 days of ad lib feeding, the onset of AA varied widely among rats. While some of the bouts were near the phase of AA observed during FD-4, other bouts appeared at very different phases (e.g. Fig. 5, #60-4, FD-5; Fig. 6, #60-8, FD-5). Since it seems unlikely that an oscillator would spontaneously produce such radical phase shifts, these bouts may be driven by a second oscillator at this phase. GENERAL DISCUSSION The results of the two experiments provide some new insights into the resetting dynamics of food-entrainable oscillators. In particular, one or two food pulses are clearly insufficient to reset the phase of the system. Although the onset of AA was delayed on the day after a phase-shifted food pulse, regardless of the magnitude and direction of the phase shift (Table 1), subsequent assessments of the phase of AA during food-deprivation probes after intervening ad lib feeding indicate that resetting had not occurred. Rather, the phase of AA appeared advanced relative to the preshift phase of AA. In Experiment l, this tendency to advance appeared already on the second and third day of food deprivation, i.e., prior to ad lib feeding. Three consecutive food pulses induced substantial phase shifts in the expected direction, which resulted in near resetting in five of eight rats in the delay conditions and in one rat in the 6-h advance condition. Four food pulses were sufficient to reset the phase of AA in all rats for which data were available. In all cases, resetting occurred when the transients had approached the new food access time prior to the ad lib condition. This implies that at least one feeding entrainable oscillator must be reentrained to the new phase in order to maintain this phase during ad lib feeding. These responses are clearly different from that of the lightentrainable oscillator. Light pulses near the onset of activity induce permanent delays, light pulses near the end of activity induce permanent advances, and light pulses during much of the inactive phase have no effect, generating the familiar phase-response curve (8). To what extent these differences are inherent in these circadian systems or are the results of procedural differences is, at this point, unclear. However, the feeding entrainable oscillators seem to have a much higher propensity for splitting than light-entrainable oscillators [c.f., (8)]. In part, the interpretation of the results hinges on the assumption that the period of the pacemaker remains reasonably stable and near 24 h in ad lib conditions. This appeared to be the case, because the phase of one bout of AA during the food deprivation probes indicated a period between 23.7 and 24.4 h. The l-day food-deprivation probes at the end of Experiment 2 also indicate free running periods near 24 h. In some rats, bouts of AA 15 or 17 days after termination of RF were within 2 h of the last entrained phase of AA, implying that the interim period of the pacemaker was 24 h _+ l0 min. However, bouts at very different phase positions were also observed, but these could be
1007 ascribed to a second oscillator (see below). The earliest studies which examined the onset of AA during food deprivation for 3-5 days immediately after R F showed only small changes (within the range of variability of entrained AA) but used a small number of subjects (2,24). However, in a study in which RF was followed by 5 days of ad lib feeding and multiple 3-day fooddeprivation probes alternating with ad lib feeding, the phase of AA appeared delayed during the first two probes, then advanced and delayed again on subsequent probes (4). Unfortunately, activity data for only one rat with SCN lesions are shown. A more recent study shows that the stability of AA in the absence of phase shifts may depend on time of day (12). When rats with SCN lesions were fed from 1430-1730 h and then food deprived after 6 days of ad lib feeding, the onset of AA was very close to that of the entrained condition. However, in rats fed from 0430-0730 h, the onset of AA during food-deprivation probes was delayed toward morning in some rats. Rather large changes in the onset of AA were observed in rats fed at 23000200 h and 0430-0730 h, but these rats had been fed previously at a different time which may have influenced the stability of phase. Since AA in these groups was displaced toward the day time, the authors speculate that external laboratory cues played a role. However, they also acknowledge that group differences and individual differences pose problems for this interpretation. In the present study, there was a tendency for AA to shift toward nighttime during ad lib conditions. The reasons for these differences are not apparent at this time. Overall, these results indicate that the stability of phase observed during food-deprivation probes may depend on a number of factors, including previous phase shifts, potential splitting of oscillators and, perhaps, external cues. It is well known that food-deprived rats are more responsive to external cues [e.g., (13)1. Another factor that could influence the phase of AA during food-deprivation probes is the time at which food access is blocked. However, in this study, no systematic relationship was observed. A previous study also reports the absence of such a relationship (12). This is not surprising, because there is some evidence that the time of food access is a more critical determinant of the phase of AA then the time of food removal (25). Both experiments produced results which are difficult to reconcile with a single-feeding entrainable oscillator, and previous studies indicate that the entrainment of AA is mediated by two (or more) circadian oscillators. The most compelling evidence is the observation of split transients (simultaneous advancing and delaying transients), especially after 8 h advances of food access (17,23). Furthermore, the ability of some rats to anticipate two meals which are presented at different periods (e.g., 24 h vs. 24.5 h) suggests the presence of two oscillators (16,22). In the second experiment reported here, a number of rats expressed bouts of AA on the second or third day of food deprivation which would require large (6 to 8 h) spontaneous phase shifts of an oscillator. This seems implausible, and an alternative hypothesis is that the phase shift dissociated two oscillators. One remained at a period near 24 h, while the other delayed with a period between 25 h and 26 h. The only direct evidence for this hypothesis is that rat #6 l - l (Fig. 2) showed a near 24-h rhythm in wheel running during ad lib feeding and a 25.8-h rhythm in drinking. The trajectories of these rhythms are shown in Fig. 2. A second oscillator would also account for the persisting AA at a previous phase observed in Experiment 1 and in the delay groups in Experiment 2. On most days, the onset of this activity was near the phase of AA at the first phase position. This persisting AA was also observed following 8-h delays in a previous study (23). The interpretation of this activity is complicated by
1008
STEPHAN
the fact that it occurred during the morning and could have been induced by laboratory noise or maintenance activities. Maintenance, indeed, triggered episodes of wheel running which usually lasted from l0 min to 1 h. These are easily seen in later stages of the experiment. However, it is unlikely that external disturbances and cues can account for this activity. First, maintenance never induced prolonged activity bouts, either prior to or after the phase shifts. Second, residual AA occurred on many weekend days, but not on all workdays, and third, the onset of AA often delayed systematically, giving the appearance of delaying transients (e.g., Fig. 1, third phase position; Fig. 2, second phase position). These transients suggest that an oscillatory process is involved. On the other hand, it is unclear what mechanism would permit an oscillator to return to a previous phase position. One possibility is that the system has the capacity to remember phase displacement. Such a mechanism would have adaptive significance in that if a periodic food source becomes available at a new time, it would be advantageous to continue to forage at the previous time because the absence of food may be temporary. However, the possibility that external cues were sufficient to entrain one oscillator cannot be ruled out entirely. The main argument against the latter hypothesis is that in the absence of resetting (i.e., after one or two food pulses), the onset of AA
during food-deprivation probes occurred at night (2200-0600 h), despite continuation of the normal laboratory routine during the intervening ad lib feeding segments. Thus, the persisting AA was expressed at the preshift phase only when a periodic food cue was also present (i.e., restricted feeding at the new phase position). Rats in the phase advance group did not show persisting activity at the preshift phase, presenting an additional argument against the external cue hypothesis. Some of these rats showed typical short activity bouts related to maintenance but not the sustained activity observed in the delay groups. The results show that the feeding entrainable pacemaker requires three to four phase-shifted food pulses to reset the phase of the system. Furthermore, the system appears to retain a memory of phase displacement which permits the expression of AA at a previous phase i f a periodic cue is present but not in its absence. ACKNOWLEDGEMENTS This work was supported by Grant No. BNS-8601821 from the National Science Foundation. The author thanks Ms. Michele Dwyer for help with the research, Ms. Sharon Wittig for secretarial assistance, Mr. Richard Brunck for help with illustrations, and Mr. Charles Badland for photography.
REFERENCES 1. Aschoff, J.; von Goetz, C.; Honma, K. Restricted feeding in rats: Effects of varying feeding cycles. Z. Tierpsychol. 63:91-11 l; 1983. 2. 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. 3. Boulos, Z.; Rusak, B. Circadian phase response curves for dark pulses in the hamster. J. Comp. Physiol. 146:411-417; 1982. 4. Clarke, J. D.; Coleman, G. J. Persistent meal-associated rhythms in sen lesioned rats. Physiol. Behav. 36:105-113; 1986. 5. 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. 6. Mrosovsky, N. Phase response curves for social entrainment. J. Comp. Physiol. 162:35-40; 1988. 7. 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-R1000; 1990. 8. Pittendrigh, C. S.; Daan, S. A functional analysis of circadian pacemakers in nocturnal rodents. IV. Entrainment: Pacemaker as a clock. J. Comp. Physiol. 106:291-331; 1976. 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. 11. 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. Sheffield, F. D.; Campbell, B. A. The role of experience in the spontaneous activity of hungry rats. J. Comp. Physiol. Psychol. 47:97100; 1954. 14. Sokolove, P. G.; Bushell, W. N. The Chi Square Periodogram, its utility for analysis of circadian rhythms. J. Tbeor. Biol. 72:131-160; 1978. 15. Stephan, F. K. Limits of entrainment to periodic feeding in rats with suprachiasmatic lesions. J. Comp. Physiol. 143:401-410; 1981. 16. Stephan, F. K. Circadian rhythm dissociation induced by periodic feeding in rats with suprachiasmatic lesions. Behav. Brain Res. 7: 81-98; 1983. 17. Stephan, F. K. Phase shifts of circadian rhythms of activity entrained to food access. Physiol. Behav. 32:663-671; 1984. 18. Stephan, F. K. The role of period and phase in interactions between feeding- and ligbt-entrainable circadian rhythms. Physiol. Behav. 36:151-158; 1986. 19. Stephan, F. K. Interaction between light- and feeding-entrainable circadian rhythms in the rat. Physiol. Bebav. 38:127-133; 1986. 20. Stephan, F. K. Coupling between feeding- and light-entrainable circadian pacemakers in the rat. Physiol. Behav. 38:537-544; 1986. 21. Stephan, F. K. Entrainment of activity to multiple feeding times in rats with suprachiasmatic lesions. Physiol. Behav. 46:489-497; 1989. 22. 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. 23. Stephan, F. K. Resetting ofa feeding-entrainable circadian clock in the rat. Physiol. Behav. 52:985-995; 1992. 24. Stephan, F. K.; Swann, J. M.; Sisk, C. L. Entrainment of circadian rhythms by feeding schedules in rats with suprachiasmatic nucleus lesions. Behav. Neural Biol. 25:545-554; 1979. 25. Stephan, F. K.; Becker, G. Entrainment of anticipatory activity to various durations of food access. Physiol. Behav. 46:731-741; 1989.
0031-9384/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
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Resetting of a Circadian Clock by Food Pulses 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 circadian clock byfood pulses. PHYSIOL BEHAV 52(5) 997-1008, 1992.--Rats with lesions of the suprachiasmatic nuclei were exposed to daily feeding until anticipatory activity (AA) developed. Meals were then phase advanced or delayed and presented for 1--4consecutive days. The phase of the circadian pacemaker was assessed during probes of total food deprivation before or after 8 days of intervening ad lib feeding. One or two food pulses caused phase delays for one cycle but were insufficientto reset the feedingentrainable pacemaker. Complete or partial resettingto 6- or 9-h advances or delays was observed in some rats after three food pulses and in all rats after four food pulses. In some rats, phase shifts of meals appeared to induce both advancing and delaying transients, and two bouts of AA appeared during food-deprivation probes. This suggests that the feeding entrainable pacemaker consists of two or more oscillators which became uncoupled after phase shifts. The persistence of AA at the preshift phase observed after initial phase delays, concomitantly with AA to the phase shifted meals, also suggests the presence of a second oscillator. Circadian rhythms
Suprachiasmaticnuclei
Phase shifts
RATS maintained on a single meal per day display an increase in activity beginning several hours prior to mealtime (9). This anticipatory activity (AA) persists in rats with lesions of the suprachiasmatic nuclei (SCN) which abolish many circadian rhythms in ad lib-fed rats, and considerable evidence indicates that AA is mediated by a circadian pacemaker that is anatomically distinct from the light-entrainable circadian pacemaker (presumably the SCN) [for review, see (10)]. The formal circadian properties of the feeding-entrainable pacemaker are best studied in rats with SCN lesions because the two circadian systems interact, albeit only weakly (18-20). Several important circadian properties of AA have been established in rats with SCN lesions. First, AA develops only when the period of meals is between 22 and 31 h (2,15,24). The upper limit of entrainment is greater if the period is increased in several steps, implying history dependence of the pacemaker (15). Second, the onset of AA relative to mealtime systematically increases with period (1), suggesting that this measure represents the phase angle of entrainment. Third, although AA rarely persists (i.e., free runs) in ad lib feeding conditions, it persists for up to 5 days during food deprivation (2). Furthermore, AA reappears during food deprivation even after weeks of intervening ad lib feeding at a phase near that of preceding entrainment (4,5,7,11). This suggests that the pacemaker free runs but that activity becomes decoupled in ad lib feeding. Fourth, following phase shifts of mealtime, several days are required until AA reentrains to the new mealtime (17,23). Transients between two steady states of entrainment are characteristic of oscillators, and can provide considerable insight into the dynamics of circadian clocks. A formal description of the effects ofa Zeitgeber on circadian oscillators can be obtained by establishing a phase-response curve (PRC) which displays the phase displacement resulting from a Zeitgeber delivered only
997
Food pulses
once at various circadian times. This technique has long been used to study light-entrainable circadian rhythms (8) and has also been used to assess the effects of dark pulses (3), as well as nonphotic signals [e.g., (6)]. As yet, no PRC for meals has been established. One of the major reasons is that AA fails to free run in ad lib feeding conditions and a state of food deprivation cannot be imposed for a sufficient number of days to use a standard protocol which assesses the effect of a single presentation of the Zeitgeber on the free-running rhythm. The present study was an attempt to study resetting behavior by a method which, while not equivalent to a standard PRC, would nevertheless provide some insight into the resetting dynamics of the feeding-entrainable circadian pacemaker. The method selected for the first experiment was to assess the phase displacement resulting from exposure to one phaseshifted signal after steady-state entrainment. Since AA is expressed during total food deprivation, the phase-shifted signal was followed by 3 days of food deprivation. Because the phase of AA might not yet have attained a steady state in 3 days, this was followed by 1 week of ad lib feeding and 3 more days of food deprivation [c.f., (4)]. The results of the first experiment indicated that a single food pulse was insufficient to reset the circadian pacemaker. Consequently, in a second experiment, the phase displacement in response to two, three, or four consecutive food pulses was assessed. METHOD
Animals, Housing, and Data Collection Twenty male Sprague-Dawley rats (body weight approximately 300 g) with SCN lesions were housed in individual soundattenuated chambers that enclosed an activity wheel with a small adjacent cage. An exhaust fan provided fresh air flow and mask-
998 ing noise. The chambers and the experimental room were maintained in constant darkness. Water was available ad lib throughout the study and bottles were refilled at 2- to 3-day intervals without opening the chambers. Food hoppers were filled with pellets (Purina Laboratory Rat Chow, No. 5001) at 3- to 5-day intervals at irregular times between 0900 and 1700 h. During replacement of bedding (weekly) and restocking of food hoppers, the door of the experimental room was left ajar. Although the brief exposure (<1 min) to low levels of light has little or no effect on the activity of rats with SCN lesions, the maintenance procedures did induce brief episodes of wheel running in some rats (see the Results and Discussion section). Wheel revolutions were recorded by means of an infrared diode and a phototransistor mounted across a tab on the wheel. Licks were recorded with contact circuits. Sixteen chambers were also equipped with contact circuits on the food hopper grid. All measures were monitored continuously by computer, and counts were stored on disk every l0 min. Food access was controlled by a motorized gate that was activated by computer.
Surgery 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. The incisor bar was 5 mm above the interaural line, and the stereotaxic coordinates were 1.3 mm anterior to bregma, +0.2 mm lateral, and 9.6 mm ventral to dura. A 1.5 mA anodal direct current was passed for 15 s through a stainless steel electrode (0.4 mm diameter) that was insulated except for 0.5 mm at the tip. A total of 24 rats received attempted SCN lesions. Drinking was monitored for 2-3 weeks in standard hanging cages, and 20 rats with the most disrupted circadian rhythms were selected for the two experiments.
Histology 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 tsm through the region of damage. Every fourth section was mounted on slides and stained with cresyl violet for light microscopic examination.
Data Analysis Event records of wheel running, drinking, and contacts with the food hopper were double plotted (48-h horizontal time scale) on a dot matrix printer. 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 to 3 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 counts over a number of consecutive days. The expression of AA during food deprivation probes was determined from event records and printouts of 10-min data counts. A bout of AA was defined by sustained activity lasting 4-8 h with pauses less than 1 h, representing a tenfold increase over activity in the preceding and succeeding 4 h. In a few cases, bouts falling short of these criteria were accepted on the basis of consistency across rats in the same group. The effect of SCN lesions on circadian rhythms was assessed with the chi-square periodogram (14).
STEPHAN PROCEDURE
Experiment 1 Four rats were placed in the activity wheels on ad lib food for 10 days. One rat died during this phase of the experiment. For the remaining three rats, food access was then limited to 2 h/day (1200-1400 h). After stable entrainment of AA, food was presented with a 4-h delay. This was followed by 3 days of food deprivation, 8 days of ad lib feeding, and 3 additional days of food deprivation. Two h/day feeding was then resumed at the new phase. When AA appeared stable, phase shifts were repeated using delays of 6, 8, and l0 h. Three, or 8 additional days ofad lib feeding, were added prior to the resumption of restricted feeding to facilitate recovery from food deprivation (see Fig. l ). A 1-h phase advance was inadvertently imposed when the computer clock was reset to standard time.
Experiment 2 Sixteen rats were placed in the activity wheels with ad lib food for 20 days. Food access was then reduced to 3 h/day for 29 days. Four rats each were then exposed to phase delays or to phase advances of mealtime of 6 or 9 h. To reduce the potential effects of noise cues from running wheels among rats, two rats exposed to phase delays were housed alternately with two rats exposed to phase advances. First, the phase shifted meal was presented for 2 consecutive days. This was followed by 8 days of ad lib feeding and 3 days of food deprivation. Restricted feeding was then resumed at the new phase position. This procedure was repeated except that the phase shifted meals were presented for three and then for four consecutive meals. During the last phase of the experiment, rats were exposed three or four times to a single day of food deprivation interspersed with ad lib feeding (see Figs. 2, 4-6). RESULTS AND DISCUSSION
Histology and Periodicity Analysis Light microscopic evaluation of the lesions indicated complete destruction oftbe SCN in the three rats used in Experiment 1. Periodicity analysis of drinking over 9 days of ad lib feeding showed no significant peaks between 4 and 30 h. Of the 16 rats used in Experiment 2, 15 brains were available for light microscopic examination. The SCN appeared totally destroyed in 13 rats. In these rats, periodicity analysis of drinking over 10 days of ad lib feeding showed no significant peaks in the circadian range, although one rat (#60-4) had uitradian peaks at 8 and 12 h and another (#60-8) at 15 h. In two rats (#61-3, #61-7) a caudal unilateral SCN remnant remained intact. Both rats had low peaks at 25 h in the periodogram. Drinking data were used for periodicity analysis because low levels of activity in some rats would have made it difficult to detect residual rhythms. The absence of free-running activity rhythms is readily apparent in the event records (Figs. 1, 2, 4-6).
Experiment 1: Effects of Single Food Pulses Several difficulties in interpreting the results must be noted at the outset. First, since the phase shifts of the Zeitgeber follow an entrained condition, the pacemaker is presumably not at its endogenous period and it is difficult to predict a priori what its period will be following a phase perturbation, i.e., the pacemaker could simply start to free run, or, because of history dependence, return to a period close to 24 h. Second, since AA rarely persists in ad lib conditions (see the Introduction section), the actual trajectory of the pacemaker to the phase of AA during food
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deprivation probes must be inferred in most cases. Over 8 days ofad lib feeding, ambiguities may arise because, for example, a period of 23.5 h would lead to the same phase as a period of 26.5 h. Third, although there is considerable evidence that the pacemaker consists of two (or more) oscillators ( 16-19,23), few rats express two separate bouts of AA during food deprivation after forced dissociation (16,22). Consequently, presumed trajectories of a second oscillator are highly subjective. Finally, it is not clear whether transients indicate the phase of the pacemaker or of a driven process, i.e., the pacemaker may be fully reset on the first cycle while the driven process may require a number of cycles to catch up, generating transients. Because the phase of the pacemaker cannot be assessed directly, the phase of AA was taken to coincide with that of the pacemaker or one of its oscillators, although this may not actually be the case. A double-plotted event record for one of the three rats in Experiment 1 is shown in Fig. 1. Prior to the first phase shift, all rats had developed AA and the onset of AA was between 0600 and 0800 h. The effect of the phase-shifted food pulse was assessed to the nearest 0.5 h by comparing the onset of AA on the day of the shift with that on the subsequent day. The onset of AA was delayed by 1.5 h in two rats and by 0.5 h in one rat (Table 1). During the subsequent 2 days of food deprivation, the onset of AA occurred earlier and preceded the preshift phase of AA by about 2 h (Fig. 1). During the second food deprivation probe, distinct bouts of activity began between 0600 and 0800 h, slightly later than on the last day of the previous food deprivation probe. This suggests that an oscillator free ran with a period >24 h during ad lib feeding. The onset of AA clearly shows that the phase of the pacemaker had not been delayed in response to the phase shifted food pulse. When restricted feeding was resumed at the new phase position, activity initially increased between 0800 and 0900 h, i.e., near the phase of AA at the preceding food deprivation probe. All three rats continued to show this activity on some days but not on others. Since maintenance was performed during the day and some rats ran in wheels while food hoppers were restocked, this induced activity contributed to the increase in activity on some days. However, maintenance-induced activity typically lasted only 10 to 60 rain, rather than 6 to 8 h. The potential influence of maintenance will be more fully discussed in Experiment 2. The presence of the early onset of AA prevented a clear assessment of the effects of the 6-h delay of mealtime. On the day after the phase shift, AA consisted of multiple dispersed bouts of activity without a distinct onset. On days 2 and 3 of food deprivation, distinct bouts of AA appeared between 0600 and 0730 h, a time comparable to that during the preceding food deprivation probe. During the second food deprivation probe after ad lib feeding, AA had advanced even more in all three rats, suggesting an oscillator period of <24 h (Fig. l, second FD after the 6-h delay). When restricted feeding was resumed, AA appeared to reentrain by delaying transients, the origin of which could be projected back to the phase of AA during food deprivation (Fig. 1). AA was absent for the initial 2 to 3 days of RF, and none of the rats achieved stable entrainment the first time the apparent transients approached mealtime. Rat M51-3 (Fig. 1) seems to show delaying transients a second time before stable AA was established. During the last 10-15 days on RF, all three rats showed fairly stable AA beginning 4-5 h prior to mealtime, as well as bouts of lower levels of activity beginning between 0700 and 1000 h. While some of these early activity bouts could have been induced by maintenance, all three rats showed early onset AA on most weekend days as well (see Experiment 2). It should be
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noted that it is highly unusual for rats with SCN lesions to require 15 days to entrain to mealtime. It seems likely that the prior phase shifts and the phase of AA during the preceding food deprivation probe was responsible for this effect. The effects of the 8-h delay pulse were also difficult to evaluate. In two rats, the major bouts of activity during food deprivation began near the phase-shifted food pulse (Fig. 1). However, since this phase was similar to that observed during the preceding food-deprivation probes, it is not likely indicative of resetting caused by the food pulse. One rat showed small dispersed bouts of activity without a clear onset. During the second food-deprivation probe after ad lib feeding, AA began between 0400 and 0600 h in the three rats, suggesting a period close to 24 h during ad lib feeding. After 8 additional days of ad lib feeding, restricted feeding was resumed and AA developed within 2-4 days. Rat M51-3 displayed an increase in activity after food access which was clearly influenced by the 1-h phase advance (Fig. 1). The effects of the 10-h delay were also difficult to ascertain. During the second and third day of food deprivation, the onset of AA occurred between 0300 and 0400 h in two rats and at 0700 h in rat M51-3 (Fig. l). During the second food-deprivation probe after ad lib feeding, the onset of AA was slightly delayed for one rat and virtually unchanged for the other two rats, indicating that the period of the pacemaker had remained near 24 h during ad lib feeding (Fig. 1). DISCUSSION These results indicate that exposure to a single-phase shifted food pulse can delay the phase of a feeding entrainable oscillator for 1 cycle (4-h delay shift), but is apparently insufficient to achieve a stable resetting of phase. The persisting activity near the initial phase of AA prevented a clear assessment of the effects of subsequent phase shifts. Apparent resetting of phase (e.g., Fig. l, 8-h delay) was most likely due to coincidence with the phase of AA during the earlier food deprivation probes. The experiment produced some unexpected results. At the second and third phase position of food access, an increase in activity occurred near the initial entrained phase of AA. While some of this activity could be attributed to maintenance, its appearance on weekends and transient like changes in its onset suggest that an endogenous oscillation may underly this activity. Furthermore, the onset of AA during the food deprivation probes was between 0300 and 0700 h, i.e., times when external disturbances are unlikely. Throughout the experiment, AA during food deprivation appeared near the initial phase of AA (Fig. 1, vertical line). During the 8 days ofad lib feeding, the phase of AA appeared relatively stable, generally advancing or delaying by less than 3
h. This indicates that the period of the oscillator was between 23.7 and 24.3 h during the 8 days ofad lib feeding. The major purpose of the second experiment was to investigate how many food pulses are required to reset the pacemaker. Because of the possibility that the unexpected results (i.e., apparent return to the preshift phase of AA) might have been influenced by the short food access time and by the food deprivation immediately after the phase shift, two changes in procedures were made. First, food access time was increased to 3 h, and the first food deprivation probe was omitted, i.e., rats were exposed to phase-shifted food pulses for 2, 3, or 4 days, with the last pulse marking the beginning of ad lib feeding for 8 days and then 2.5 or 3.5 days of food deprivation were imposed to assess the phase of AA. Food deprivation always began 12 h away from the phase-shifted mealtime. RESULTS
Phase Delay l--Two Pulses Representative event records for two rats each in the 6-h and 9-h delay condition are shown in Figs. 2 and 4. All rats displayed AA prior to the first phase shift. The effects of the phase-shifted food pulse were assessed by measuring (to the nearest 0.5 h) the onset of AA on the day of the shift and on the subsequent day (since the animals had not yet been exposed to the second pulse, the effect on the phase of the oscillator is like that in Experiment 1). The onset of AA was delayed by 1.5 h for all four rats exposed to the 6-h delay and by l to 2 h for rats exposed to the 9-h delay (Table l). After 8 days of ad lib feeding, the phase of AA was assessed during 3 days of food deprivation. In the 6-h delay group, the phase of AA on the first day of deprivation was close to that of the preshift phase of AA. On the next day, short bouts of activity occurred 6 to 8 h earlier and then delayed over the next 2 days (Fig. 2, FD-2). Rat #61-1 showed increased levels of activity during ad lib feeding, with a period close to 24 h which suggests the trajectory of an oscillator to the phase of AA on the first day of food deprivation. However, it seems rather unlikely that an oscillator would then advance by as much as 8 h in a single cycle, only to delay over the subsequent two cycles. The examination of drinking records showed an increased tendency to lick at a period of about 26 h. Consequently, the expression of AA during food deprivation could have resulted from splitting of two oscillators, one of which remained near the phase (and period) of the preshift phase of AA, while the other delayed. This explanation would also account for the onset of AA following resumption of restricted feeding at the new phase position, i.e., daily delays give the appearance of delaying transients (Fig. 2). During the next 26 days on restricted feeding, two of four rats in the 6-h delay group showed increased levels of activity
1002
STEPHAN
near the preshift phase of AA. As was the case in Experiment 1, this activity occurred during daytime hours (0800-1800 h) and could have been influenced by external cues. However, when the average distribution of activity on Saturdays and Sundays over 3 consecutive weekends was compared with that of the 2 preceding work days, the early onset of activity was present or absent in both (Fig. 3). In the 9-h delay group, two of the rats displayed a short bout of activity (at 0200 or 0400 h) on the first day of food deprivation (Fig. 4, #60-5, FD-1) which could have resulted from delaying transients (c.f., Fig. 2). In the third rat (Fig. 4, #61-6, FD-1), the bout appeared near the preshift phase, and in the fourth rat, activity was too dispersed to determine the onset of AA. During the next 3 days, the onset of AA for all four rats was between 0600 and 0800 h, near or earlier than the preshift phase of AA. Upon resumption of restricted feeding, a mechanical food gate failure rendered the data of rat #61-5 useless for 10 days. The other three rats showed elevated activity near the preshift phase and only low levels of AA to food access for 10-20 days (Fig. 4). As in the 6-h delay group, some of this persisting activity
could have been associated with maintenance, but it also occurred on weekends. AA to food access became more pronounced in the latter stages at this phase position, and at least in rat #61-6 (Fig. 4), this coincided with apparent delaying transients from the persisting AA. Clearly, two food pulses were not sufficient to reset the phase of AA after 6- or 9-h delays.
Phase Delay 2--Three Pulses Three food pulses were sufficient to elicit delaying transients in all four rats in the 6-h delay group (Fig. 2). During the food deprivation probe, the onset of AA varied widely among the four rats (Fig. 2, FD-2). For rat #61-1, AA began near the phase shifted food access time, indicating a possible resetting of one oscillator. However, in the other three rats, the onset of AA seemed unrelated to either the new or to the preshift phase of AA. Rather, AA appeared near the time of the previous food deprivation probe (e.g., Fig. 2, #60-2, FD-1, and FD-2). When restricted feeding resumed, three rats reestablished AA by the 2nd day. Rat #61-1 (Fig. 2) showed splitting of AA with 2 days of advancing and 3 days of delaying transients.
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1004 In the 9-h delay group, one rat showed delaying transients, but two rats displayed AA to the second and third pulse without intervening transients (e.g., Fig. 4, #61-6). Low levels of activity prevented a clear assessment of transients in the other rat (Fig. 4, 60-5). The phase of AA during the food deprivation probe was near the new phase position in all four rats, indicating a possible reset and a free running period only slightly longer than 24 h during ad lib feeding (Fig. 4, FD-2). AA was quickly reestablished when restricted feeding was resumed. These results indicate that three food pulses were sufficient to induce a change in phase of AA large enough to qualify as resetting in five of eight rats after 6or 9-h phase delays.
Phase Delay 3--Four Pulses Prior to the third phase shift, all rats displayed stable AA and did not show any obvious residual effects of previous phase shifts. Most of the short bouts of activity between 0900 and 1600 h were related to maintenance. All rats in both groups showed daily delays to the four food pulses, and in seven of eight rats the onset of AA on day 4 was close to that expected for reentrainment (Figs. 2 and 4). In one rat, AA became dispersed into multiple bouts over some 14 h preceding food access (not shown). During food deprivation, the phase of AA in the 6-h delay group was near the new phase position (Fig. 2, FD-3) indicating that the phase of at least one oscillator had been reset by the four food pulses and that its period had remained close to 24 h during ad lib feeding. Rat #6 l- l (Fig. 3) showed enhanced activity during ad lib feeding, supporting this interpretation. AA was quickly reestablished when restricted feeding resumed, presumably because the phase of one oscillator was already near that of the new food access time. This indicates that in order to achieve a 6-h delay, four food pulses are sufficient to reset the phase of the system. In the 9-h delay group, rat M60-6 showed only dispersed activity. In two rats, the phase of AA was near the new phase on the first day but was advanced by some 6 h on the next 2 days, i.e., near the preshift phase of AA (Fig. 4, #61-6, FD-3). This may indicate splitting, because it is unlikely that a single oscillator would phase advance 6 h in one cycle. The fourth rat had very low activity counts due to a rubbing activity wheel (Fig. 4, #60-5). All four rats reestablished AA within a few days.
STEPHAN
Phase Advance 1--Two Pulses All rats displayed AA prior to the first phase shift. The effect of advancing mealtime on the phase of AA was assessed by comparing the onset of AA on the day prior to the phase shift with that on the day of the phase shift (because the Zeitgeber occurred prior to the entrained AA). All rats in both groups except #618 (Fig. 6) showed a delayed onset of AA on the day of the shift (Table 1), and none of the rats anticipated the second food pulse. During 3 days of food deprivation, the onset of activity was advanced compared to the preshift phase of AA in all rats (Figs. 5 and 6, FD-l). Because similar advances were observed in the phase delay groups, this response may not be due to partial resetting. After the resumption of restricted feeding, all but one rat (#61-3, Fig. 5) displayed AA within 3 days. Some rats showed brief bursts of activity after food access every 3-5 days which were associated with maintenance in most cases (e.g., Fig. 6, #61-8).
Phase Advance 2--Three Pulses The data for rat #61-4 in the 6-h phase advance group could not be used for the remainder of the experiment due to equipment failure. In the remaining rats, the phase advance again induced delays in the onset of AA on the day of the shift (Figs. 5 and 6). The magnitude of the delay was generally comparable to that induced by the first phase shift. On the next day, four rats showed further delays in AA (e.g., Fig. 6, #60-8). The three rats in the 6-h advance group showed AA to the third phase shifted pulse (Fig. 5), but no AA was observed in the 9-h advance group. During food deprivation, a complex pattern of AA was observed. In all rats, two bouts of AA occurred, in some cases on the same day (e.g., Fig. 6, #61-8, FD-2), or at very different phase positions on day 1 and clay 2 of deprivation (Fig. 5, #604; Fig. 6, #60-8, FD-2). The two bouts suggests splitting of two oscillators, and this interpretation is reinforced by apparent split transients upon resumption of restricted feeding in rat #60-8 (Fig. 6). AA was quickly reestablished in all seven rats when restricted feeding was resumed, but appeared much less stable in the 9-h advance group than in the 6-h advance group.
Phase Advance--Four Pulses Food Deprivation Probes Without Phase Shifts Due to a programming error, rats in the 6-h delay group were not exposed to food deprivation on the second day after restricted feeding was ended. During the 3 single days of food deprivation alternating with ad lib feeding, three rats showed AA at a phase close to the previous entrained phase of AA (Fig. 2, #60-2, #6 ll, FD-5, and FD-6). This seems to indicate that in the absence of phase shifts the period of at least one oscillator remains close to 24 h during ad lib feeding so that the phase changes observed at the preceding two food deprivation probes were due to resetting. However, in three of the four rats, a second bout of activity occurred at a different phase position. In rat #6 l-1 (Fig. 2), increased activity during ad lib feeding appears to converge on this second bout. The different phase positions of the two bouts could be accounted for by splitting. In the 9-h delay group, activity bouts during the four food deprivation probes appeared near or slightly in advance of the last entrained phase of AA in all four rats, indicating oscillator periods near 24 h (Fig. 4, FD-4, to FD-7).
On the day of the phase shift all rats showed a delayed onset of AA. The three rats in the 6-h advance group displayed AA to the second and subsequent food pulses with daily advancing transients (Fig. 5). Rats in the 9-h advance group anticipated the third or fourth pulse (Fig. 6). This indicates that the phase oftbe pacemaker, or at least one of its oscillators, had been reset in two to three cycles in the 6-h advance group and in three to four cycles in the 9-h advance group. As in previous studies (17,23), the advancing transients in AA seem to be masked by food access (i.e., AA is expressed only when its onset precedes food access). During ad lib feeding, rat #60-8 again showed enhanced levels of activity with a period of about 24.4 h, suggesting a possible trajectory of an oscillator (Fig. 6). On the first day of food deprivation, the phase of AA, compared to the day of the last food pulse, was delayed by 0.5 to 4 h, suggesting that the period of at least one oscillator remained near 24 h in all seven rats. The phase of AA on the second day remained fairly stable in most rats, but several rats showed a marked advance on subsequent days (e.g., Fig. 5, #60-4, FD-3), suggesting instability or splitting
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RESETTING BY FOOD PULSES despite the apparent resetting. The data for one rat (#60-3) became unusable at this stage because of mechanical problems with the activity wheel. AA was reestablished quickly when R F was resumed. Rat #60-4 showed delaying transients from the advanced bout of AA (Fig. 6). All remaining rats consistently anticipated food access until RF was terminated.
Food Deprivation Probes Without Phase Shifts During the first food deprivation probe, the onset of AA was at or slightly earlier than on the last day of restricted feeding in all six rats (Figs. 5 and 6, FD-4). However, during the three lday deprivation probes following 10 days of ad lib feeding, the onset of AA varied widely among rats. While some of the bouts were near the phase of AA observed during FD-4, other bouts appeared at very different phases (e.g. Fig. 5, #60-4, FD-5; Fig. 6, #60-8, FD-5). Since it seems unlikely that an oscillator would spontaneously produce such radical phase shifts, these bouts may be driven by a second oscillator at this phase. GENERAL DISCUSSION The results of the two experiments provide some new insights into the resetting dynamics of food-entrainable oscillators. In particular, one or two food pulses are clearly insufficient to reset the phase of the system. Although the onset of AA was delayed on the day after a phase-shifted food pulse, regardless of the magnitude and direction of the phase shift (Table 1), subsequent assessments of the phase of AA during food-deprivation probes after intervening ad lib feeding indicate that resetting had not occurred. Rather, the phase of AA appeared advanced relative to the preshift phase of AA. In Experiment l, this tendency to advance appeared already on the second and third day of food deprivation, i.e., prior to ad lib feeding. Three consecutive food pulses induced substantial phase shifts in the expected direction, which resulted in near resetting in five of eight rats in the delay conditions and in one rat in the 6-h advance condition. Four food pulses were sufficient to reset the phase of AA in all rats for which data were available. In all cases, resetting occurred when the transients had approached the new food access time prior to the ad lib condition. This implies that at least one feeding entrainable oscillator must be reentrained to the new phase in order to maintain this phase during ad lib feeding. These responses are clearly different from that of the lightentrainable oscillator. Light pulses near the onset of activity induce permanent delays, light pulses near the end of activity induce permanent advances, and light pulses during much of the inactive phase have no effect, generating the familiar phase-response curve (8). To what extent these differences are inherent in these circadian systems or are the results of procedural differences is, at this point, unclear. However, the feeding entrainable oscillators seem to have a much higher propensity for splitting than light-entrainable oscillators [c.f., (8)]. In part, the interpretation of the results hinges on the assumption that the period of the pacemaker remains reasonably stable and near 24 h in ad lib conditions. This appeared to be the case, because the phase of one bout of AA during the food deprivation probes indicated a period between 23.7 and 24.4 h. The l-day food-deprivation probes at the end of Experiment 2 also indicate free running periods near 24 h. In some rats, bouts of AA 15 or 17 days after termination of RF were within 2 h of the last entrained phase of AA, implying that the interim period of the pacemaker was 24 h _+ l0 min. However, bouts at very different phase positions were also observed, but these could be
1007 ascribed to a second oscillator (see below). The earliest studies which examined the onset of AA during food deprivation for 3-5 days immediately after R F showed only small changes (within the range of variability of entrained AA) but used a small number of subjects (2,24). However, in a study in which RF was followed by 5 days of ad lib feeding and multiple 3-day fooddeprivation probes alternating with ad lib feeding, the phase of AA appeared delayed during the first two probes, then advanced and delayed again on subsequent probes (4). Unfortunately, activity data for only one rat with SCN lesions are shown. A more recent study shows that the stability of AA in the absence of phase shifts may depend on time of day (12). When rats with SCN lesions were fed from 1430-1730 h and then food deprived after 6 days of ad lib feeding, the onset of AA was very close to that of the entrained condition. However, in rats fed from 0430-0730 h, the onset of AA during food-deprivation probes was delayed toward morning in some rats. Rather large changes in the onset of AA were observed in rats fed at 23000200 h and 0430-0730 h, but these rats had been fed previously at a different time which may have influenced the stability of phase. Since AA in these groups was displaced toward the day time, the authors speculate that external laboratory cues played a role. However, they also acknowledge that group differences and individual differences pose problems for this interpretation. In the present study, there was a tendency for AA to shift toward nighttime during ad lib conditions. The reasons for these differences are not apparent at this time. Overall, these results indicate that the stability of phase observed during food-deprivation probes may depend on a number of factors, including previous phase shifts, potential splitting of oscillators and, perhaps, external cues. It is well known that food-deprived rats are more responsive to external cues [e.g., (13)1. Another factor that could influence the phase of AA during food-deprivation probes is the time at which food access is blocked. However, in this study, no systematic relationship was observed. A previous study also reports the absence of such a relationship (12). This is not surprising, because there is some evidence that the time of food access is a more critical determinant of the phase of AA then the time of food removal (25). Both experiments produced results which are difficult to reconcile with a single-feeding entrainable oscillator, and previous studies indicate that the entrainment of AA is mediated by two (or more) circadian oscillators. The most compelling evidence is the observation of split transients (simultaneous advancing and delaying transients), especially after 8 h advances of food access (17,23). Furthermore, the ability of some rats to anticipate two meals which are presented at different periods (e.g., 24 h vs. 24.5 h) suggests the presence of two oscillators (16,22). In the second experiment reported here, a number of rats expressed bouts of AA on the second or third day of food deprivation which would require large (6 to 8 h) spontaneous phase shifts of an oscillator. This seems implausible, and an alternative hypothesis is that the phase shift dissociated two oscillators. One remained at a period near 24 h, while the other delayed with a period between 25 h and 26 h. The only direct evidence for this hypothesis is that rat #6 l - l (Fig. 2) showed a near 24-h rhythm in wheel running during ad lib feeding and a 25.8-h rhythm in drinking. The trajectories of these rhythms are shown in Fig. 2. A second oscillator would also account for the persisting AA at a previous phase observed in Experiment 1 and in the delay groups in Experiment 2. On most days, the onset of this activity was near the phase of AA at the first phase position. This persisting AA was also observed following 8-h delays in a previous study (23). The interpretation of this activity is complicated by
1008
STEPHAN
the fact that it occurred during the morning and could have been induced by laboratory noise or maintenance activities. Maintenance, indeed, triggered episodes of wheel running which usually lasted from l0 min to 1 h. These are easily seen in later stages of the experiment. However, it is unlikely that external disturbances and cues can account for this activity. First, maintenance never induced prolonged activity bouts, either prior to or after the phase shifts. Second, residual AA occurred on many weekend days, but not on all workdays, and third, the onset of AA often delayed systematically, giving the appearance of delaying transients (e.g., Fig. 1, third phase position; Fig. 2, second phase position). These transients suggest that an oscillatory process is involved. On the other hand, it is unclear what mechanism would permit an oscillator to return to a previous phase position. One possibility is that the system has the capacity to remember phase displacement. Such a mechanism would have adaptive significance in that if a periodic food source becomes available at a new time, it would be advantageous to continue to forage at the previous time because the absence of food may be temporary. However, the possibility that external cues were sufficient to entrain one oscillator cannot be ruled out entirely. The main argument against the latter hypothesis is that in the absence of resetting (i.e., after one or two food pulses), the onset of AA
during food-deprivation probes occurred at night (2200-0600 h), despite continuation of the normal laboratory routine during the intervening ad lib feeding segments. Thus, the persisting AA was expressed at the preshift phase only when a periodic food cue was also present (i.e., restricted feeding at the new phase position). Rats in the phase advance group did not show persisting activity at the preshift phase, presenting an additional argument against the external cue hypothesis. Some of these rats showed typical short activity bouts related to maintenance but not the sustained activity observed in the delay groups. The results show that the feeding entrainable pacemaker requires three to four phase-shifted food pulses to reset the phase of the system. Furthermore, the system appears to retain a memory of phase displacement which permits the expression of AA at a previous phase i f a periodic cue is present but not in its absence. ACKNOWLEDGEMENTS This work was supported by Grant No. BNS-8601821 from the National Science Foundation. The author thanks Ms. Michele Dwyer for help with the research, Ms. Sharon Wittig for secretarial assistance, Mr. Richard Brunck for help with illustrations, and Mr. Charles Badland for photography.
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13. Sheffield, F. D.; Campbell, B. A. The role of experience in the spontaneous activity of hungry rats. J. Comp. Physiol. Psychol. 47:97100; 1954. 14. Sokolove, P. G.; Bushell, W. N. The Chi Square Periodogram, its utility for analysis of circadian rhythms. J. Tbeor. Biol. 72:131-160; 1978. 15. Stephan, F. K. Limits of entrainment to periodic feeding in rats with suprachiasmatic lesions. J. Comp. Physiol. 143:401-410; 1981. 16. Stephan, F. K. Circadian rhythm dissociation induced by periodic feeding in rats with suprachiasmatic lesions. Behav. Brain Res. 7: 81-98; 1983. 17. Stephan, F. K. Phase shifts of circadian rhythms of activity entrained to food access. Physiol. Behav. 32:663-671; 1984. 18. Stephan, F. K. The role of period and phase in interactions between feeding- and ligbt-entrainable circadian rhythms. Physiol. Behav. 36:151-158; 1986. 19. Stephan, F. K. Interaction between light- and feeding-entrainable circadian rhythms in the rat. Physiol. Bebav. 38:127-133; 1986. 20. Stephan, F. K. Coupling between feeding- and light-entrainable circadian pacemakers in the rat. Physiol. Behav. 38:537-544; 1986. 21. Stephan, F. K. Entrainment of activity to multiple feeding times in rats with suprachiasmatic lesions. Physiol. Behav. 46:489-497; 1989. 22. 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. 23. Stephan, F. K. Resetting ofa feeding-entrainable circadian clock in the rat. Physiol. Behav. 52:985-995; 1992. 24. Stephan, F. K.; Swann, J. M.; Sisk, C. L. Entrainment of circadian rhythms by feeding schedules in rats with suprachiasmatic nucleus lesions. Behav. Neural Biol. 25:545-554; 1979. 25. Stephan, F. K.; Becker, G. Entrainment of anticipatory activity to various durations of food access. Physiol. Behav. 46:731-741; 1989.