BEHAVIORAL A N D N E U R A L BIOLOGY 2 5 , 5 4 5 - - 5 5 4
(1979)
Entrainment of Circadian Rhythms by Feeding Schedules in Rats with Suprachiasmatic Lesions FRIEDRICH K . S T E P H A N , J E N N I F E R M . S W A N N , AND CHERYL L . SISK t
Department of Psychology, Florida State University, Tallahassee, Florida 32306 Rats with lesions of the suprachiasmatic nucleus (SCN) and controls were maintained in constant light and exposed to a restricted feeding schedule at 23and 24-hr intervals, as well as to a 12-hr phase shift in the feeding schedule. Despite the absence of circadian periodicity in acitivity or drinking in ad lib. conditions, rats with SCN lesions showed anticipatory wheel running to both feeding schedules, comparable to sham-operated rats. Following the 12-hr phase shift, transients qualitatively similar to those seen following phase shifts in the light-dark cycle were observed. During a 3-day period of total food deprivation following prolonged entrainment to a 24-hr feeding schedule, wheel running persisted with a near 24-hr periodicity while return to ad lib. conditions resulted in a rapid desynchronization of activity. These results indicate that anticipatory wheel running in rats with SCN lesions is based on endogenous circadian oscillators which are entrainable by feeding schedules in the circadian range. Apparently such oscillators free run under certain conditions (food deprivation) but become rapidly desynchronized in others (ad lib. feeding). The evidence strongly supports a multioscillator model of the circadian system in mammals.
Rats exposed to restricted feeding schedules at 24-hr intervals substantially increase wheel running activity several hours prior to food availability (e.g., Richter, 1927; Bolles & Stokes, 1965; Edmonds & Adler, 1977a). Since rats appear to be unable to anticipate 19- or 29-hr feeding schedules, it seems reasonable to assume that anticipatory wheel running is based on an endogenous circadian rhythm (Bolles & Stokes, 1965). Because of the importance of the suprachiasmatic nucleus (SCN) in the generation and entrainment of circadian rhythms (for review see Zucker, Rusak, & King, 1976; Block & Page, 1978; Menaker, Takahashi, & Eskin, 1978), we have recently studied the ability of rats with SCN lesions to anticipate 18- and 24-hr feeding schedules in constant light (Stephan, Swann, & Sisk, 1979). Rats with SCN lesions were able to anticipate 24-hr schedules as well as controls but both groups were unable 1 This research was supported in part by Psychobiology Training Grant MH-I1218 from the National Institute of Mental Health to Dr. D. R. Kenshalo, Florida State University. 545 0163-1047/79/040545-10502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
546
STEPHAN, SWANN, AND SISK
to anticipate an 18-hr schedule. Furthermore, Krieger, Hauser, and Krey (1977) reported entrainment of corticosterone and body temperature rhythms to 24-hr feeding schedules in rats with SCN lesions. Such results strongly support a multi-oscillator model of circadian control (c.f. Moore-Ede, Schmelzer, Kass, & Herd, 1976). The apparent entrainment of activity, corticosterone and temperature in rats with SCN lesions suggest not only the presence of additional oscillators but also their ability to entrain to periodic environmental cues. This interpretation, if correct, would be extremely helpful in the experimental analysis of the circadian system. While the available evidence is consistent with this model, it is not sufficiently conclusive to rule out alternative explanations. Thus far, the strongest evidence that anticipatory wheel running in rats with SCN lesions is based on circadian oscillations is the lack of entrainment to an 18-hr feeding schedule (Stephan et al., 1979). This seems to rule out gastrointestinal (or other) feedback cues with arbitrary time constants as anticipatory stimuli. Entrainment to a 24-hr schedule, on the other hand, might result from unknown or unobserved environmental cues which may be present even in highly controlled laboratory conditions. The demonstration of entrainment to a feeding schedule in the circadian range (but not exactly 24 hr) would minimize this possibility. Edmonds and Adler (1977b) showed anticipatory wheel running in intact rats to a 25-hr schedule in constant light. However, the period of the free-running rhythm in this case can be expected to nearly coincide with the feeding schedule. Perhaps the most critical observation is that anticipatory wheel running appears only in the presence of an entraining stimulus; the activity of rats with SCN lesions quickly becomes desynchronized in ad lib. conditions, so that no "free-running" rhythm is evident. This suggests that circadian oscillators outside SCN are unable to maintain synchrony without an external periodic cue. Alternatively, the possibility that anticipatory wheel running is based on an hourglass mechanism which requires resetting on a daily basis must also be considered. The present experiment was designed to seek additional evidence for the hypothesis that anticipatory wheel running is based on endogenous circadian oscillations. First, rats with SCN lesions maintained in constant light were exposed to a 23-hr feeding schedule to exclude the role of subtle external cues with 24-hr periodicity and also, for intact rats, to force entrainment to a period shorter than the free running period expected in constant light. Second, following entrainment to a 24-hr feeding schedule, a 12-hr phase shift was imposed. The transients following such a shift can also provide clues as to the involvement of circadian rhythms. Third, following prolonged exposure to a 24-hr feeding schedule, rats were food deprived for 3 days to determine whether periodic anticipatory wheel
FEEDING SCHEDULES ENTRAIN CIRCADIAN RHYTHMS
547
running persists after removal of the entraining stimulus. This effect may have been obscured by return to ad lib. conditions in the earlier study (Stephan et al., 1979). METHOD Adult male Sprague-Dawley rats were randomly assigned to control and SCN lesion groups. Surgical procedures were performed under chloropent (Fort Dodge Lab., Inc., Iowa; 0.3 ml/100 g) anesthesia. S C N lesions. Tungsten electrodes, insulated except for 0.2 mm at the tip, were aimed at the SCN. With the incisor bar 5.0 mm above the interaural line, the coordinates were 1.5 mm anterior to bregma, ___0.2mm lateral, and 8.4 mm below the dura. A 2 mA anodal DC was passed for 20 see.
Controls. No surgical procedures were performed on these rats. During a 2-week recovery period all rats were housed in standard hanging cages on a 12:12 LD cycle (lights on 0800). Water intake was read daily at 0800 and 2000 hr. Four rats with SCN lesions were selected on the basis of nearly equal light- and dark-period water intakes. (During the last 5 days these rats consumed an average of 49.4, 52.5, 53.1, and 53.4% of their daily water in the dark period.) These rats and four randomly chosen controls (percentage dark to total intakes over 5 days 83.8, 89.4, 90.4, and 93.2) were then placed in activity wheels with a small adjacent housing compartment. Each activity unit was enclosed in a sound attenuated chamber. An exhaust fan provided a constant fresh air flow as well as a masking noise. Shielded 15-W light bulbs provided constant illumination throughout the experiment (approximately 6 lux at the center of the cage). Licks were monitored by drinkometers consisting of infrared lightemitting diodes and phototransducers mounted in front of water spouts. The running wheels activated one microswitch closure per revolution. Both measures were monitored continuously on event records at a paper speed of 45.7 cm/day. Records were assembled on cardboard for inspection. For photographic reproduction, it was necessary to retouch pen deflections with black ink. All rats were exposed to the following procedures: 1. Baseline. Eight days with food and water ad lib. 2. Twenty-three hr schedule. Food was removed at 0800 and replaced from 2200 to 2400. Food was then available at 23-hr intervals for a total of 15 access periods of 2 hr each. Water was ad lib. throughout the experiment. 3. Twenty-four hr schedule. Food was available from 0800 to 1000 hr for 8 consecutive days. On the ninth day, the access period was shifted by 12 hr (2000 to 2200 hr). This was continued for 14 days. 4. Food deprivation. No food was available for 3 consecutive days. 5. A d lib. feeding. Food was ad lib. for the remainder of the experiment.
548
STEPHAN, SWANN, AND SISK
Histology. At the conclusion of the experiment, rats with SCN lesions were anesthetized with sodium pentobarbital, perfused through the heart with saline followed by 10% formalin. Frozen sections were cut at 40/zm through the suprachiasmatic region, mounted on glass slides and stained with cresyl violet. Data analysis. After visual inspection of the event records, a modification of Enright's periodogram (1965) recently suggested by Sokolove and Bushell (1978), was used to detect periodicity during the 8-day baseline period and the last 10 days of the 23-hr feeding schedule. Activity was quantified from the event records by cumulating activity counts in hourly intervals. For sustained bursts of activity which were beyond resolution of individual pen deflections, 1 rpm was counted. Although this procudure results in clipping of the data (range of 0-60 counts per hour), periodicity analysis is not seriously affected unless noise levels are high (Sokolove & Bushell, 1978). The sampling range was from 18 to 30 hr at 0.2-hr intervals. Significance of peaks in the periodogram was assessed by using critical values of X2p.1 with ~ set at 0.001 or 0.005. When corrected for repeated sampling at hourly intervals, the actual probability of false peaks is approximately 0.01 or 0.06, respectively. Periodicity during the 24-hr feeding schedules was so marked in the event records that the periodogram analysis was deemed unnecessary. RESULTS Microscopic examination of the brain sections showed that both SCN were completely destroyed in all four rats. Since the lesions were intentionally made large, there was substantial damage to the optic chiasm, anterior hypothalamic area and the retro-chiasmatic region. A representative lesion is shown in Fig. 1.
Baseline Period Periodogram analysis showed no significant peaks in the activity records of SCN rats, supporting the absence of circadian periodicity in activity or drinking apparent on visual inspection of event records (see Fig. 3). (A number of minor peaks in the periodogram suggested the presence of ultradian frequencies.) In contrast, three control rats showed significant peaks (p < .001) at 25.0, 25.0, and 24.6 hr (see Fig. 4). One control rat had a peak at 24.8 hr which failed to reach significance. This rat had unusually low levels of baseline activity (20-40 revolutions/day); however, event records of drinking clearly indicated a free running rhythm with a period of approximately 25 hr.
Twenty-three Hour Feeding Schedule Three to five days after the initiation of the 23-hr feeding schedule, a marked increase in activity preceded food access by 1-5 hr for all SCN
F E E D I N G S C H E D U L E S E N T R A I N CIRCADIAN RHYTHMS
549
aS N
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550
STEPHAN, SWANN, AND SISK
and control animals. Periodograms for SCN rats showed a significant peak at 23.0 hr for three rats and at 23.2 hr for one rat (p < .001 for two rats and p < .005 for the other two). A second peak appeared in the periodogram at 26-29 hr as a result of the bimodal activity pattern shown in Fig. 2. This figure also indicates that one activity peak preceded food access and was not simply due to passive driving. Periodograms for controls were considerably more complex with only one rat having a narrow peak at 23 hr. The peaks of the other controls were broader with peaks at 23.6, 23.6, and 24.6 hr. Event records suggested the presence of a free-running rhythm with periods longer than 24 hr in addition to the 23-hr anticipatory activity, the periodogram method is only moderately successful in resolving two frequencies, in particular if the initial phase difference is small (Sokolove & Bushell, 1978).
Twenty-four Hour Feeding Schedule Anticipatory activity to the 24-hr schedule was easily identifiable in the event records of all rats. Of considerable interest was the observation that the onset of anticipatory activity appeared to adhere to a near 23-hr period for 1-3 days (not counting the first day of the 24-hr schedule). The 12-hr phase shift in the feeding schedule produced a gradual shift in the bouts of anticipatory activity, somewhat like the transients following a phase shift in the LD cycle. Although the variability in the onset of anticipatory activity made it difficult to ascertain the number of days required for entrainment, it was clear that a number of cycles were required before entrainment to the phase-shifted feeding schedule was reasonably stable
18 o
CONTROL
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(1979)
Entrainment of Circadian Rhythms by Feeding Schedules in Rats with Suprachiasmatic Lesions FRIEDRICH K . S T E P H A N , J E N N I F E R M . S W A N N , AND CHERYL L . SISK t
Department of Psychology, Florida State University, Tallahassee, Florida 32306 Rats with lesions of the suprachiasmatic nucleus (SCN) and controls were maintained in constant light and exposed to a restricted feeding schedule at 23and 24-hr intervals, as well as to a 12-hr phase shift in the feeding schedule. Despite the absence of circadian periodicity in acitivity or drinking in ad lib. conditions, rats with SCN lesions showed anticipatory wheel running to both feeding schedules, comparable to sham-operated rats. Following the 12-hr phase shift, transients qualitatively similar to those seen following phase shifts in the light-dark cycle were observed. During a 3-day period of total food deprivation following prolonged entrainment to a 24-hr feeding schedule, wheel running persisted with a near 24-hr periodicity while return to ad lib. conditions resulted in a rapid desynchronization of activity. These results indicate that anticipatory wheel running in rats with SCN lesions is based on endogenous circadian oscillators which are entrainable by feeding schedules in the circadian range. Apparently such oscillators free run under certain conditions (food deprivation) but become rapidly desynchronized in others (ad lib. feeding). The evidence strongly supports a multioscillator model of the circadian system in mammals.
Rats exposed to restricted feeding schedules at 24-hr intervals substantially increase wheel running activity several hours prior to food availability (e.g., Richter, 1927; Bolles & Stokes, 1965; Edmonds & Adler, 1977a). Since rats appear to be unable to anticipate 19- or 29-hr feeding schedules, it seems reasonable to assume that anticipatory wheel running is based on an endogenous circadian rhythm (Bolles & Stokes, 1965). Because of the importance of the suprachiasmatic nucleus (SCN) in the generation and entrainment of circadian rhythms (for review see Zucker, Rusak, & King, 1976; Block & Page, 1978; Menaker, Takahashi, & Eskin, 1978), we have recently studied the ability of rats with SCN lesions to anticipate 18- and 24-hr feeding schedules in constant light (Stephan, Swann, & Sisk, 1979). Rats with SCN lesions were able to anticipate 24-hr schedules as well as controls but both groups were unable 1 This research was supported in part by Psychobiology Training Grant MH-I1218 from the National Institute of Mental Health to Dr. D. R. Kenshalo, Florida State University. 545 0163-1047/79/040545-10502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
546
STEPHAN, SWANN, AND SISK
to anticipate an 18-hr schedule. Furthermore, Krieger, Hauser, and Krey (1977) reported entrainment of corticosterone and body temperature rhythms to 24-hr feeding schedules in rats with SCN lesions. Such results strongly support a multi-oscillator model of circadian control (c.f. Moore-Ede, Schmelzer, Kass, & Herd, 1976). The apparent entrainment of activity, corticosterone and temperature in rats with SCN lesions suggest not only the presence of additional oscillators but also their ability to entrain to periodic environmental cues. This interpretation, if correct, would be extremely helpful in the experimental analysis of the circadian system. While the available evidence is consistent with this model, it is not sufficiently conclusive to rule out alternative explanations. Thus far, the strongest evidence that anticipatory wheel running in rats with SCN lesions is based on circadian oscillations is the lack of entrainment to an 18-hr feeding schedule (Stephan et al., 1979). This seems to rule out gastrointestinal (or other) feedback cues with arbitrary time constants as anticipatory stimuli. Entrainment to a 24-hr schedule, on the other hand, might result from unknown or unobserved environmental cues which may be present even in highly controlled laboratory conditions. The demonstration of entrainment to a feeding schedule in the circadian range (but not exactly 24 hr) would minimize this possibility. Edmonds and Adler (1977b) showed anticipatory wheel running in intact rats to a 25-hr schedule in constant light. However, the period of the free-running rhythm in this case can be expected to nearly coincide with the feeding schedule. Perhaps the most critical observation is that anticipatory wheel running appears only in the presence of an entraining stimulus; the activity of rats with SCN lesions quickly becomes desynchronized in ad lib. conditions, so that no "free-running" rhythm is evident. This suggests that circadian oscillators outside SCN are unable to maintain synchrony without an external periodic cue. Alternatively, the possibility that anticipatory wheel running is based on an hourglass mechanism which requires resetting on a daily basis must also be considered. The present experiment was designed to seek additional evidence for the hypothesis that anticipatory wheel running is based on endogenous circadian oscillations. First, rats with SCN lesions maintained in constant light were exposed to a 23-hr feeding schedule to exclude the role of subtle external cues with 24-hr periodicity and also, for intact rats, to force entrainment to a period shorter than the free running period expected in constant light. Second, following entrainment to a 24-hr feeding schedule, a 12-hr phase shift was imposed. The transients following such a shift can also provide clues as to the involvement of circadian rhythms. Third, following prolonged exposure to a 24-hr feeding schedule, rats were food deprived for 3 days to determine whether periodic anticipatory wheel
FEEDING SCHEDULES ENTRAIN CIRCADIAN RHYTHMS
547
running persists after removal of the entraining stimulus. This effect may have been obscured by return to ad lib. conditions in the earlier study (Stephan et al., 1979). METHOD Adult male Sprague-Dawley rats were randomly assigned to control and SCN lesion groups. Surgical procedures were performed under chloropent (Fort Dodge Lab., Inc., Iowa; 0.3 ml/100 g) anesthesia. S C N lesions. Tungsten electrodes, insulated except for 0.2 mm at the tip, were aimed at the SCN. With the incisor bar 5.0 mm above the interaural line, the coordinates were 1.5 mm anterior to bregma, ___0.2mm lateral, and 8.4 mm below the dura. A 2 mA anodal DC was passed for 20 see.
Controls. No surgical procedures were performed on these rats. During a 2-week recovery period all rats were housed in standard hanging cages on a 12:12 LD cycle (lights on 0800). Water intake was read daily at 0800 and 2000 hr. Four rats with SCN lesions were selected on the basis of nearly equal light- and dark-period water intakes. (During the last 5 days these rats consumed an average of 49.4, 52.5, 53.1, and 53.4% of their daily water in the dark period.) These rats and four randomly chosen controls (percentage dark to total intakes over 5 days 83.8, 89.4, 90.4, and 93.2) were then placed in activity wheels with a small adjacent housing compartment. Each activity unit was enclosed in a sound attenuated chamber. An exhaust fan provided a constant fresh air flow as well as a masking noise. Shielded 15-W light bulbs provided constant illumination throughout the experiment (approximately 6 lux at the center of the cage). Licks were monitored by drinkometers consisting of infrared lightemitting diodes and phototransducers mounted in front of water spouts. The running wheels activated one microswitch closure per revolution. Both measures were monitored continuously on event records at a paper speed of 45.7 cm/day. Records were assembled on cardboard for inspection. For photographic reproduction, it was necessary to retouch pen deflections with black ink. All rats were exposed to the following procedures: 1. Baseline. Eight days with food and water ad lib. 2. Twenty-three hr schedule. Food was removed at 0800 and replaced from 2200 to 2400. Food was then available at 23-hr intervals for a total of 15 access periods of 2 hr each. Water was ad lib. throughout the experiment. 3. Twenty-four hr schedule. Food was available from 0800 to 1000 hr for 8 consecutive days. On the ninth day, the access period was shifted by 12 hr (2000 to 2200 hr). This was continued for 14 days. 4. Food deprivation. No food was available for 3 consecutive days. 5. A d lib. feeding. Food was ad lib. for the remainder of the experiment.
548
STEPHAN, SWANN, AND SISK
Histology. At the conclusion of the experiment, rats with SCN lesions were anesthetized with sodium pentobarbital, perfused through the heart with saline followed by 10% formalin. Frozen sections were cut at 40/zm through the suprachiasmatic region, mounted on glass slides and stained with cresyl violet. Data analysis. After visual inspection of the event records, a modification of Enright's periodogram (1965) recently suggested by Sokolove and Bushell (1978), was used to detect periodicity during the 8-day baseline period and the last 10 days of the 23-hr feeding schedule. Activity was quantified from the event records by cumulating activity counts in hourly intervals. For sustained bursts of activity which were beyond resolution of individual pen deflections, 1 rpm was counted. Although this procudure results in clipping of the data (range of 0-60 counts per hour), periodicity analysis is not seriously affected unless noise levels are high (Sokolove & Bushell, 1978). The sampling range was from 18 to 30 hr at 0.2-hr intervals. Significance of peaks in the periodogram was assessed by using critical values of X2p.1 with ~ set at 0.001 or 0.005. When corrected for repeated sampling at hourly intervals, the actual probability of false peaks is approximately 0.01 or 0.06, respectively. Periodicity during the 24-hr feeding schedules was so marked in the event records that the periodogram analysis was deemed unnecessary. RESULTS Microscopic examination of the brain sections showed that both SCN were completely destroyed in all four rats. Since the lesions were intentionally made large, there was substantial damage to the optic chiasm, anterior hypothalamic area and the retro-chiasmatic region. A representative lesion is shown in Fig. 1.
Baseline Period Periodogram analysis showed no significant peaks in the activity records of SCN rats, supporting the absence of circadian periodicity in activity or drinking apparent on visual inspection of event records (see Fig. 3). (A number of minor peaks in the periodogram suggested the presence of ultradian frequencies.) In contrast, three control rats showed significant peaks (p < .001) at 25.0, 25.0, and 24.6 hr (see Fig. 4). One control rat had a peak at 24.8 hr which failed to reach significance. This rat had unusually low levels of baseline activity (20-40 revolutions/day); however, event records of drinking clearly indicated a free running rhythm with a period of approximately 25 hr.
Twenty-three Hour Feeding Schedule Three to five days after the initiation of the 23-hr feeding schedule, a marked increase in activity preceded food access by 1-5 hr for all SCN
F E E D I N G S C H E D U L E S E N T R A I N CIRCADIAN RHYTHMS
549
aS N
e~
~t
550
STEPHAN, SWANN, AND SISK
and control animals. Periodograms for SCN rats showed a significant peak at 23.0 hr for three rats and at 23.2 hr for one rat (p < .001 for two rats and p < .005 for the other two). A second peak appeared in the periodogram at 26-29 hr as a result of the bimodal activity pattern shown in Fig. 2. This figure also indicates that one activity peak preceded food access and was not simply due to passive driving. Periodograms for controls were considerably more complex with only one rat having a narrow peak at 23 hr. The peaks of the other controls were broader with peaks at 23.6, 23.6, and 24.6 hr. Event records suggested the presence of a free-running rhythm with periods longer than 24 hr in addition to the 23-hr anticipatory activity, the periodogram method is only moderately successful in resolving two frequencies, in particular if the initial phase difference is small (Sokolove & Bushell, 1978).
Twenty-four Hour Feeding Schedule Anticipatory activity to the 24-hr schedule was easily identifiable in the event records of all rats. Of considerable interest was the observation that the onset of anticipatory activity appeared to adhere to a near 23-hr period for 1-3 days (not counting the first day of the 24-hr schedule). The 12-hr phase shift in the feeding schedule produced a gradual shift in the bouts of anticipatory activity, somewhat like the transients following a phase shift in the LD cycle. Although the variability in the onset of anticipatory activity made it difficult to ascertain the number of days required for entrainment, it was clear that a number of cycles were required before entrainment to the phase-shifted feeding schedule was reasonably stable
18 o
CONTROL
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