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Memory for feeding time: Possible dependence on coupled circadian oscillators
Physiology &Behavior, Vol. 32, pp. 25-30. Copyright ©Pergamon Press Ltd., 1984. Printed in the U.S.A.
0031-9384/84 $3.00 + .00
Memory for Feeding Time: Possible Dependence on Coupled Circadian Oscillators A L A N M. R O S E N W A S S E R ,
R O D N E Y J. P E L C H A T
AND NORMAN
T. A D L E R
Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104 R e c e i v e d 27 M a y 1983 ROSENWASSER, A. M., R. J. PELCHAT AND N. T. ADLER. Memory for feeding time: Possible dependence on coupled circadian oscillators. PHYSIOL BEHAV 32(1) 25-30, 1984.--Rats maintained on limited-access daily feeding schedules develop food-anticipatory activity rhythms which coexist with the photic circadian activity rhythm. These food anticipatory rhythms aplbear to depend upon a food-entrainable circadian oscillator which is separate and distinct from the light-entrainable circadian oscillator system. This study explored the long-term behavior of the putative food-entrainable oscillator in the presence and in the absence of a feeding schedule, and under light-dark cycles and constant light. The results suggest that a food-entrainable oscillator can show persisting self-sustained oscillations in the absence of a feeding schedule, and that the food- and light-entrainable circadian oscillators may show varying degrees of coupling, depending upon feeding conditions. Such a flexible coupling arrangement may allow the oscillator system to function as a "continuously consulted clock" in the adaptive temporal coordination of behavior with stable and unstable environmental periodicities. Circadian rhythms
Wheel running
Food restriction
RICHTER [20] demonstrated that rats fed during a restricted period at the same time each day show "'anticipatory" wheel-running activity preceding each daily access to food. Bolles and coworkers [1,3] later showed that food anticipatory activity rhythms (FARs) depend upon a circadian timing system: FARs were seen under 24 hour (circadian), but not 19 or 29 hour (non-circadian) feeding schedules. Subsequent work on FARs has attempted to further elucidate the circadian properties of these rhythms, and the nature of the oscillator(s) which underlies them. This work has shown that circadian feeding schedules are capable of synchronizing food anticipatory rhythms in a number of species, and in a variety of behavioral and physiological parameters (cf., [12,27]; also see [4] for a review). Despite the circadian properties of FARs, the oscillator underlying them seems to be both functionally and anatomically distinct from the oscillator(s) responsible for photically influenced activity rhythms. FARs may be observed concurrently with light-entrained or free-running " p h o t i c " activity rhythms, even when the two rhythms have different periods [5, 7, 8, 9]. In addition, FARs are not disrupted by lesions of the suprachiasmatic nuclei (SCN), a putative circadian " m a s t e r oscillator" for photically influenced circadian rhythms (see [21] for a review). Such lesions abolish photic activity rhythms, while sparing FARs, in the same animal [5, 13, 18, 22, 25, 26]. The sparing of FARs by SCN lesions provides the strongest evidence, in the rat, for
Entrainment
non-SCN circadian oscillators, and supports the view that overall circadian organization depends upon a multioscillator circadian timing system (see discussion in [14], pp. 185-186). However, like other circadian rhythms, FARs occur only within a limited circadian "range of entrainment" (that is, when the period of the feeding schedule is close to 24 hours) [1, 3, 5, 22, 25]. Furthermore, under certain conditions, FARs appear to be at least somewhat self-sustaining: when a feeding schedule is terminated and the animal is then maintained under continuous food deprivation, daily increases in activity are seen for five or more days at the projected time of the previously scheduled feeding. On the other hand, when the feeding schedule is replaced by continuous ad lib food access, persistence of F A R s is more variable, generally less robust, and damps out more rapidly than under deprivation [5,26] (but see [7] for an exception). While even the temporary persistence of FARs in the absence of a feeding schedule provides strong evidence for an endogenous circadian oscillator underlying FARs, it is not yet known whether the temporary persistence and gradual damping of FARs reflects the properties of the underlying oscillator. In other words, the damping of the overt FAR may result from the damping of the oscillator, or may be due to the uncoupling of the overt rhythm from a persisting underlying oscillator. Indeed, there are several mechanisms by which such uncoupling may occur. First, the behavioral expression of the
~A preliminary report of these data was presented at the annual meeting of the Eastern Psychological Association, Baltimore, MD, April 1982 (A. M. Rosenwasser and N. T. Adler, Long-term effects of circadian feeding schedules). These studies were supported in part by NIH grant HD 04522 (to NTA), NSF grant BNS 81-20816 (to NTA) and NIH Postdoctoral Fellowship AM 06394 (to AMR).
25
26
ROSENWASSER,
FAR may be abolished by an "'extinction" process in which food availability is no longer differentially paired with time of day. In this view, the expression of the FAR is essentially a temporally conditioned response, where food access serves as the unconditioned stimulus and time of day as the temporal conditioned stimulus. Of course, this conditioning hypothesis requires the involvement of a circadian timing mechanism in the recognition of the time-of-day conditioned stimulus, since FARs are not seen when the period of the feeding schedule deviates too far from 24 hours. Second, there are motivational factors which may directly affect the overt expression of the FAR and thereby obscure the behavior of the underlying oscillator. For example, under ad lib conditions the expression of the FAR may be abolished due to the loss of food motivation which accompanies restoration of normal levels of food intake and body weight (in those studies which report food intakes, feeding schedules have been shown to reduce daily intake to about 75% of ad lib levels; e.g., [5]). In addition, under deprivation conditions, prolonged starvation may lead directly to non-specific decreases in activity levels. The purpose of the present experiments was to determine whether long-term persistence of FARs could be uncovered in the absence of a circadian feeding schedule. Whatever the mechanism of the damping of FARs under such conditions, it is clearly not possible to determine the long-term operating characteristics of the food-anticipatory oscillator from the limited persistence of the overt FAR which follows the cessation of the feeding schedule. The demonstration of longterm persistent FARs would strengthen the evidence for a self-sustaining, food-entrainable circadian oscillator in the rat, and permit the analysis of its free-running behavior as well as potential coupling relationships between the two circadian systems (photic and food-entrainable).
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Subjects and Apparatus Eighteen male albino Sprague-Dawley rats (Charles River), weighing approximately 300 g at the start of the experiments, were maintained in modified Wahmann running wheels. The side cage of each wheel was removed and replaced by an automated rotary feeding device which moved powdered rat chow in and out of the reach of the animal. The animal was therefore confined to the wheel compartment. The feeding device consisted of 4 food cups, each of which held more than 2 day's diet. When a feeding schedule was in effect, a fresh food cup was presented to the animal for 2 hours each day. Under ad lib, the (partially depleted) food cup was replaced by a fresh cup at the same time each day, thus providing continuous access to fresh food. Water was freely available throughout the experiments from a standard bottle mounted on the wall near the feeder• The running wheels were housed three to a cabinet, one per shelf. A single 25 W incandescent light was mounted on each shelf of the cabinet, and each cabinet had an independently programmable lighting schedule. Each revolution of the wheel was recorded on an Esterline-Angus event recorder• These records were cut into 24 hour strips and then pasted on boards with each day's record below the previous day, to construct standard circadian actograms. The cabinets were entered about once per week to replenish food and water supplies and to provide routine maintainance. This was done under dim red light if the cabinet was scheduled for darkness at that time.
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FIG. 1. Wheel running record from a representative group 1 animal. Successive days are plotted from top to bottom on the y-axis and time of day (24-hour span) on the x-axis. The animal was maintained throughout the experiment under a light-dark cycle with the dark segment indicated by the black bar above the record. During the period marked "'A," a restricted feeding schedule was in effect which allowed two hours of food access each day beginning at the vertical line in the record. During the period marked "B," the animal was exposed to complete food deprivation. At all other times, free access to food was allowed.
Procedures In an earlier study, Bolles and Moot [2] noted that an FAR which had damped out during ad lib feeding could be rapidly reinstated by reimposition of food deprivation; however, they did not formally present the results of this procedure. This observation suggested the use of the " m e m o r y " paradigm described below as a means of uncovering persisting rhythmicity of the food-entrainable oscillator after the complete damping of the overt FAR. During an initial baseline period of at least 2 weeks (Phase 1), the animals were permitted ad lib access to food while they acclimated to the wheels and the operation of the feeder device. In Phase 2, the animals were maintained under a 24 hr feeding schedule for at least 20 days. The ad lib baseline and the feeding schedule were both conducted under LD 14:10 (lights on at 2100 hr), and the feeding schedule provided 2 hrs of food access each day near the middle of the
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FIG. 2. Wheel running record from a representative group 2 animal. The record is "double plotted" on the time-of-day axis (48-hour span) to facillitate inspection of phase-shifting pattern. On the last day of the feeding schedule (period "A") the light-dark cycle was phase-shifted (10-hour delay). The initial light-dark cycle is represented above the record, and the shifted light-dark cycle below the record. The vertical line indicates the beginning of food access under the feeding schedule. In addition, a dotted line has been drawn anchored to the onset of food access on the last day of the feeding schedule and (visually) parallel to the onset of the phase-shifting, light-entrained activity rhythm. This procedure was intended to extrapolate the phase of the previous feeding schedule, relative to the pbotic rhythm, across the ad lib period. As in Fig. 1, the period marked "B" indicates complete food deprivation.
light period (0400-0600 hr). In Phase 3, the animals were returned to ad lib feeding; on the last day of the feeding schedule, the food became available at the usual time and then remained available continuously thereafter. During the second ad lib period, six animals remained under the same LD cycle (group 1), six were subjected to a 10 hr delay phase-shift of the LD cycle on the first day of ad lib (group 2), and six were maintained under constant light (LL) beginning on the first ad lib day (group 3). At various intervals after the beginning of Phase 3 (7 to 50 days), the animals in all groups were completely food deprived for four to seven days, and then returned to ad lib. The periods of deprivation began at different times of day for the different animals in order to rule out the possibility that running activity was triggered by the time of food removal. In addition, some animals in group 3 were subjected to repeated periods of deprivation, with each period separated from the others by at least three days of intervening ad lib feeding.
R E S U L T S
Representative results from one animal in each group are shown in the figures (Fig. 1, group 1; Fig. 2, group 2; Fig. 3, group 3). The results from all animals were quite similar and will be described together. As is typical of the nocturnal rat under LD and ad lib feeding conditions, wheel running in Phase 1 was mainly confined to the dark segment of the LD cycle. When the food access schedule was imposed (Phase 2), all animals developed FARs: robust wheel running occurred in the hours preceding each daily feeding. These FARs developed gradually over the 3-7 days after the introduction of the feeding schedule. In addition to the occurrence of FARs, the photic (nocturnal) wheel running rhythm sometimes showed changes during Phase 2; these included reduced magnitude and/or transient phase disruptions (usually advances). In other animals, the nocturnal activity rhythm was unaffected
ROSENWASSER, PELCHAI'
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FIG. 3. Wheel running record from a representative group 3 animal. On the last day of the feeding schedule, the light-dark cycle indicated above the record was replaced by constant light (LL). "B," "'C." and "'D" indicate successive periods of complete food deprivation. Other conventions as in Fig. 2.
by the feeding schedule. Despite these differences, all animals showed two daily components of running activity during Phase 2, one during the dark and one in anticipation of feeding. At the beginning of Phase 3 (return to ad lib), the FARs damped quickly; the animals showed 0-2 days of persisting anticipatory activity. In addition, the photic rhythms responded in typical fashion to the different manipulations of the lighting schedule in the different groups: animals in group 1 continued to show their normal nocturnal running rhythm,
animals in group 2 phase-shifted to the new LD phase in about one week, and animals in group 3 showed free-running activity rhythms with greater-than-24 hr periods under LL. The clarity and persistence of free-running rhythms in group 3 varied considerably, which is typical of rat activity rhythms under LL. The periods of deprivation imposed during Phase 3 occurred many days after the complete damping of overt foodanticipatory activity. Nevertheless, all animals in group ! showed robust running activity at the time of the previously
C I R C A D I A N MEMORY
29
scheduled daily feeding. This " m e m o r y " for feeding time could be obtained as many as 50 days after the termination of the original feeding schedule and the beginning of ad lib feeding; no longer intervals were tested. Similarly, animals in groups 2 an 3 showed robust deprivation-induced running at the time that feeding would have occurred under the previous feeding schedule, relative to the now shifted (group 2) or free-running (group 3) photic rhythm. In other words, all animals in all groups showed deprivation-induced running which began about 8--9 hours before the onset of the photic activity rhythm, the same phase relation which prevailed between the two rhythms under the original feeding schedule (we would expect the running induced during the deprivation tests to be approximately centered at the extrapolated time of food onset, as is seen during deprivation tests which immediately follow the termination of a feeding schedule; cf., [5]). In some animals, the deprivation-induced activity developed over 2-3 days, while in others, it seemed to persist for 2-3 days after the end of the deprivation test and the return to ad lib. In general, it appeared that deprivation-induced activity developed more slowly and was abolished more quickly with relatively longer intervening ad lib periods. However, all animals showed the induced activity at all tested intervals. The results with repeated memory tests in group 3 animals were more variable: In some animals, the occurrence of deprivation-induced activity became less robust with each test, and/or could not be elicited during the third or later tests. The animal shown in Fig. 3 showed a bout of activity at the appropriate phase relative to the free-running photic activity rhythm during the first two memory tests, but the deprivation-induced increase in activity seemed to occur too early in the circadian cycle during the third memory test, 75 days after the termination of the original feeding schedule. It is possible that this result was due to the extreme damping of the photic free-running rhythm seen by this point in the record. Thus the animals in all groups appeared to " l e a r n " the circadian phase of food availability during the feeding schedule, to " r e m e m b e r " the phase of food access over many days of intervening ad lib feeding, and to " e x p e c t " to be fed at the appropriate time, relative to the photic rhythm, when subsequently subjected to complete deprivation. DISCUSSION
In this study, rats demonstrated an ability to remember the relative circadian phase (of the photic rhythm) at which feeding occurred under a previous daily food access schedule. These results are in good agreement with those of two recent studies which appeared while this report was in preparation. Coleman et al. [6] have presented results from a paradigm very similar to our group 3, and also report that the photic and food-entrainable oscillators seem to free-run in parallel under ad lib access to food. In addition, they also presented one case in which dissociation between the two rhythms occurred (see Fig. 3 in the present report). The conditions under which such dissociations occur cannot be specified at present. In addition, Mori et al. [15] have recently shown that persisting food-related drinking rhythms may free-run in parallel with the photic drinking rhythm under ad lib feeding conditions. While it may be possible to explain the present results with reference to only a single light-entrainable oscillator, previous studies [5, 6, 7, 8, 18, 22, 25, 26] strongly support
the idea that F A R ' s depend on a separate, food-entrainable circadian oscillator. Thus we suggest that the ability to remember the relative phase of the feeding schedule, as shown in the present study, may require the involvement of both a food-entrainable circadian oscillator as well as the classical, photically entrainable circadian oscillator. In this scheme, the phase of the food-entrainable oscillator is controlled by the feeding schedule while it is in effect. However, during the subsequent ad lib conditions the food-entrainable oscillator continues to operate, but is controlled by (coupled to) the photic oscillator. On the other hand, overt anticipatory behavior is expressed only under conditions of relative or complete food deprivation or body weight reduction (i.e., during the feeding schedule, during the initial few days of ad lib feeding, and during the deprivation tests). This model of the rat's memory for feeding time requires that the coupling relationship between the food- and lightentrainable oscillators be dependent on environmental conditions, in particular, the presence or absence of a feeding schedule. This seems plausible, since it is well known that 24-hr food-anticipatory rhythms can exist concurrently with non-24-hr free-running photic rhythms under L L [5,9]. We therefore suggest that under a feeding schedule, the foodentrainable oscillator is entrained by the feeding schedule and is "'uncoupled" from the photic oscillator, while under ad lib feeding, the food-entrainable oscillator becomes coupled to and phase shifts or free-runs in parallel with the photic oscillator. Such a flexible coupling relationship between circadian oscillators may allow animals to synchronize their behavioral activity ("foraging") with both geophysically stable and predictable environmental periodicities (i.e., LD cycles) as well as unstable environmental periodicities (i.e., food availability). This coupled oscillator system may function as a "continuously consulted clock" (cf., [19]) in the adaptive temporal coordination of behavior. (It seems most likely that the SCN is a critical anatomical locus for the photic oscillator, while recent work suggests that the ventromedial nucleus (VMH) may be critical for the expression of FARs [ 10,11]. Neural connections which could potentially mediate coupling between these two nuclei have been demonstrated both anatomically and electrophysiologically [16,23].) Our hypothesis seems to further require that the two oscillators be capable of stable coupling at a n y mutual phase relationship during ad lib; a rigorous test of this hypothesis would require replicating this study using various different food access times during the feeding schedule, since in the present study all animals were " t r a i n e d " with the same phase relationship between the food access and light-dark schedules. It is also interesting to note that the phase of the food-entrainable oscillator appears not to be influenced by the spontaneous feeding rhythm which occurs in phase with the photic activity rhythm under ad lib conditions. Of course, the spontaneous feeding rhythm is of much lower amplitude than the " f o r c e d " feeding rhythm which occurs under a feeding schedule, and may simply be too weak a stimulus to entrain the food-entrainable oscillator. Additional evidence is available to further suggest that the two oscillators may interact through " w e a k " coupling mechanisms. Changes in the phase (under LD) or period (under LL) of the photic activity rhythm have been seen following the introduction and removal of a feeding schedule [5, 7, 9], and " b e a t " phenomena have been reported to occur under L L when the two rhythms periodically overlap [5]. Furthermore, a recent report [24] has suggested that the strength of interaction between the two concurrently ob-
3(I
R()SENWASSER,
s e r v e d r h y t h m s d e p e n d s upon the similarity of t h e i r periods o r upon their mutual p h a s e relationship, as predicted by oscillator theory (cf., " M a g n e t E f f e c t s " I28]; see also 117] for an a l t e r n a t i v e model which m a k e s similar p r e d i c t i o n s a b o u t coupling strength). T h e s e results d e m o n s t r a t e the effects of the feeding schedule, or the f o o d - e n t r a i n a b l e oscillator, on the photic activity r h y t h m . H o w e v e r , in the present report we d e m o n s t r a t e that the photic s y s t e m m a y influence the b e h a v i o r of the F A R , at least during ad lib feeding c o n d i t i o n s w h e n the two s y s t e m s seem to p h a s e shift or free-run in parallel. T h e r e f o r e , we t e n t a t i v e l y suggest that a mutual coupling relationship exists b e t w e e n t h e s e two oscillator systems. A n o t h e r r e c e n t s t u d y s u p p o r t s the h y p o t h e s i s that the f o o d - a n t i c i p a t o r y oscillator m a y be c a p a b l e of l o n g - t e r m self-sustained r h y t h m s . S t e p h a n [22] has s h o w n a p p a r e n t f r e e - r u n n i n g F A R s in S C N - l e s i o n e d a n i m a l s m a i n t a i n e d on feeding s c h e d u l e s j u s t outside the circadian range of e n t r a i n m e n t . While S t e p h a n ' s data m a y show persisting
PI£I.CHAI" \ND
AI)I3iR
F A R s in the p r e s e n c e of a Cnon-entraining) food s c h e d u l e and in the a b s e n c e of the photic oscillator, our m e m o r y paradigm d e m o n s t r a t e s persisting F A R s in the p r e s e n c e o f the photic oscillator but in the a b s e n c e of a feeding schedule, It would t h e r e f o r e be of interest to repeat the m e m o r y experi m e n t s in S C N - l e s i o n e d animals, in an a t t e m p t to e x a m i n e the " t r u e " free-running characteristics o f the FAR oscillator. In s u m m a r y , the results of the present sludy, when considered along with previous work, support the following c o n c l u s i o n s : t l) Feeding cycles e n t r a i n a circadian oscillator w h i c h c o n t i n u e s to o p e r a t e after the t e r m i n a t i o n o f the feeding s c h e d u l e , regardless o f i n t e r v e n i n g ad lib feeding conditions: (2) U n d e r ad lib, this oscillator free-runs or p h a s e shifts in parallel with the photic oscillator, i.e., they are c o u p l e d ; ~3) T h e b e h a v i o r a l e x p r e s s i o n of the lboda n t i c i p a t o r y oscillator requires c o n d i t i o n s of at least rehttive food d e p r i v a t i o n (or body weight loss): i4) T h e p h a s e of the f o o d - a n t i c i p a t o r y oscillator is not influenced by the spontaneous feeding r h y t h m which o c c u r s u n d e r ad lib conditions.
REFERENCES
1. Bolles, R. C. and J. DeLorge. The rat's adjustment to a-diurnal feeding cycles. J Comp Physiol Psychol. 55: 760-762. 1962. 2. Bolles, R. C. and S. A. Moot. The rat's anticipation of two meals a day. J Comp Physiol Psychol 83: 510-514, 1973. 3. Bolles, R. C. and L. W. Stokes. Rat's anticipation of diurnal and a-diurnal feeding. J Comp Physiol Psychol 69: 290-294, 1965. 4. Boulos, Z. and M. Terman. Food availability and daily biological rhythms. Neuros'ei Biobehav Rev 4:11%131, 1980. 5. Boulos, Z., A. M. Rosenwasser and M. Terman. Feeding schedules and the circadian organization of behavior in the rat. Behav Brain Res I: 39-65, 1980. 6. Coleman, G. J., S. Harper, J. D. Clarke and S. Armstrong. Evidence for a separate meal-associated oscillator in the rat. Physiol Behav 29:107-115, 1982. 7. Edmonds, S. C. and N. T. Adler. Food and light as entrainers of circadian running activity in the rat. Physiol Behav 18: 915919, 1977. 8. Edmonds, S. C. and N. T. Adler. The multiplicity of biological oscillators in the control of circadian running activity in the rat. Physiol Behav 18: 921-930, 1977. 9. Gibbs, F. P. Fixed interval feeding does not entrain the circadian pacemaker in blind rats. Am J Physiol 236: R249-R253, 1979. 10. Inouye, S.-I. T. Ventromedial hypothalamic lesions eliminate anticipatory activities of restricted daily feeding schedules in the rat. Brain Res 250: 183-187, 1982. 11. Krieger, D. T. Ventromedial hypothalamic lesions abolish food-shifted circadian adrenal and temperature rhythmicity. Endocrinology 106: 64%654, 1980. 12. Krieger, D. T. and H. Hauser. Comparison of synchronization of circadian corticosteroid rhythms by photoperiod and food. Proc Natl Acad Sci USA 75: 1577-1581, 1978. 13. Krieger, D. T., H. Hauser and L. C. Krey. Suprachiasmatic nuclear lesions do not abolish food-shifted circadian adrenal and temperature rhythmicity. Science 197: 398-399, 1977. 14. Moore-Ede, M. C., F. M. Sulzman and C. A. Fuller. The Clocks that Time Us. Cambridge, MA: Harvard Press, 1982. 15. Mori, T., K. Nagai and H. Nakagawa. Dependence of memory of meal time upon circadian biological clock in rats. Physiol Behav 30: 259-265, 1983.
16. Oomura, Y., T. ()no. H. Nishino. H. Kita, N. Shimizu, S. ishizuka and K. Sasaki. Hypothalamic control of feeding behavior: Modulation by the suprachiasmatic nucleus. In: Biological Rhythms and their Central Mechanism, edited by M. Suda, O. Hayaishi and H. Nakagawa. Amsterdam: Elsevier/North Holland, 1979, pp. 295-308. 17. Pavlidis, T. Spatial and temporal organization of populations of interacting oscillators. In: The Molecular BasL~ o f Circadian Rhythms. edited by J. W. Hastings and H. G. Schweiger. Berlin: Dahlem Konferenzen, 1976, pp. 131-148. 18. Phillips, J. L. M. and P. J. Mikulka. Effects of restricted ff)od access upon locomotor activity in rats with suprachiasmatic nucleus lesions. Phvsiol Behav 23: 257-262, 1979. 19. Pittendrigh, C. S. Perspectives in the study of bioh)gical clocks. In: Perspectives in Marine Biology, edited by A. A. BuzzatiTraverso. Berkeley: University of California Press, 1958. pp. 239-268. 20. Richter, C. P. A behavioristic study of the activity of the rat. Comp Psyehol Monogr 1: 1-55, 1922. 21. Rusak, B. and I. Zucker, Neural regulation of circadian rhythms. Physiol Rev 59: 449-526, 1979. 22. Stephan, F. K. Limits of entrainment to periodic feeding in rats with suprachiasmatic lesions..I (otnp Phvsiol 143: 401-410, 1981. 23. Stephan, F. K., K. Berkley and R. Moss. Efferent connections of the rat suprachiasmatic nucleus. Neuroscience 6: 2625-2641, 1981. 24. Stephan, F. K., J. A. Donaldson and A. Robbins. Food and light as entrainers of circadian rhythms. Soc Neuro~ci Abstr 7: 857, 1981. 25. Stephan, F. K., J. M. Swarm and C. L, Sisk. Anticipation of 24-hour feeding schedules in rats with lesions of the suprachiasmatic nucleus. Behav Neural Biol 25: 346-363, 1979. 26. Stephan, F. K., J. M. Swarm and C. L, Sisk. Entrainment of circadian rhythms by feeding schedules in rats with suprachiasmatic lesions. Behav Neural Biol 25: 545-554, 1979. 27. Sulzman, F. M., C. A. Fuller and M. C. Moore-Ede. Comparison of synchronization of primate circadian rhythms by light and food. Am J Physiol 243: R130-R135, 1978. 28. Von Holst, E. Die relative koordination als phanomen und als methods zentralnervoser funktionsanalyse. Ergeb Physiol Biol Chem 43: 228, 1939. Translated in The Behavioral Physiology qf Animals and Man, vol l, translated by R. Martin. Coral Gables: University Miami Press, 1973, pp. 33-135.
0031-9384/84 $3.00 + .00
Memory for Feeding Time: Possible Dependence on Coupled Circadian Oscillators A L A N M. R O S E N W A S S E R ,
R O D N E Y J. P E L C H A T
AND NORMAN
T. A D L E R
Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104 R e c e i v e d 27 M a y 1983 ROSENWASSER, A. M., R. J. PELCHAT AND N. T. ADLER. Memory for feeding time: Possible dependence on coupled circadian oscillators. PHYSIOL BEHAV 32(1) 25-30, 1984.--Rats maintained on limited-access daily feeding schedules develop food-anticipatory activity rhythms which coexist with the photic circadian activity rhythm. These food anticipatory rhythms aplbear to depend upon a food-entrainable circadian oscillator which is separate and distinct from the light-entrainable circadian oscillator system. This study explored the long-term behavior of the putative food-entrainable oscillator in the presence and in the absence of a feeding schedule, and under light-dark cycles and constant light. The results suggest that a food-entrainable oscillator can show persisting self-sustained oscillations in the absence of a feeding schedule, and that the food- and light-entrainable circadian oscillators may show varying degrees of coupling, depending upon feeding conditions. Such a flexible coupling arrangement may allow the oscillator system to function as a "continuously consulted clock" in the adaptive temporal coordination of behavior with stable and unstable environmental periodicities. Circadian rhythms
Wheel running
Food restriction
RICHTER [20] demonstrated that rats fed during a restricted period at the same time each day show "'anticipatory" wheel-running activity preceding each daily access to food. Bolles and coworkers [1,3] later showed that food anticipatory activity rhythms (FARs) depend upon a circadian timing system: FARs were seen under 24 hour (circadian), but not 19 or 29 hour (non-circadian) feeding schedules. Subsequent work on FARs has attempted to further elucidate the circadian properties of these rhythms, and the nature of the oscillator(s) which underlies them. This work has shown that circadian feeding schedules are capable of synchronizing food anticipatory rhythms in a number of species, and in a variety of behavioral and physiological parameters (cf., [12,27]; also see [4] for a review). Despite the circadian properties of FARs, the oscillator underlying them seems to be both functionally and anatomically distinct from the oscillator(s) responsible for photically influenced activity rhythms. FARs may be observed concurrently with light-entrained or free-running " p h o t i c " activity rhythms, even when the two rhythms have different periods [5, 7, 8, 9]. In addition, FARs are not disrupted by lesions of the suprachiasmatic nuclei (SCN), a putative circadian " m a s t e r oscillator" for photically influenced circadian rhythms (see [21] for a review). Such lesions abolish photic activity rhythms, while sparing FARs, in the same animal [5, 13, 18, 22, 25, 26]. The sparing of FARs by SCN lesions provides the strongest evidence, in the rat, for
Entrainment
non-SCN circadian oscillators, and supports the view that overall circadian organization depends upon a multioscillator circadian timing system (see discussion in [14], pp. 185-186). However, like other circadian rhythms, FARs occur only within a limited circadian "range of entrainment" (that is, when the period of the feeding schedule is close to 24 hours) [1, 3, 5, 22, 25]. Furthermore, under certain conditions, FARs appear to be at least somewhat self-sustaining: when a feeding schedule is terminated and the animal is then maintained under continuous food deprivation, daily increases in activity are seen for five or more days at the projected time of the previously scheduled feeding. On the other hand, when the feeding schedule is replaced by continuous ad lib food access, persistence of F A R s is more variable, generally less robust, and damps out more rapidly than under deprivation [5,26] (but see [7] for an exception). While even the temporary persistence of FARs in the absence of a feeding schedule provides strong evidence for an endogenous circadian oscillator underlying FARs, it is not yet known whether the temporary persistence and gradual damping of FARs reflects the properties of the underlying oscillator. In other words, the damping of the overt FAR may result from the damping of the oscillator, or may be due to the uncoupling of the overt rhythm from a persisting underlying oscillator. Indeed, there are several mechanisms by which such uncoupling may occur. First, the behavioral expression of the
~A preliminary report of these data was presented at the annual meeting of the Eastern Psychological Association, Baltimore, MD, April 1982 (A. M. Rosenwasser and N. T. Adler, Long-term effects of circadian feeding schedules). These studies were supported in part by NIH grant HD 04522 (to NTA), NSF grant BNS 81-20816 (to NTA) and NIH Postdoctoral Fellowship AM 06394 (to AMR).
25
26
ROSENWASSER,
FAR may be abolished by an "'extinction" process in which food availability is no longer differentially paired with time of day. In this view, the expression of the FAR is essentially a temporally conditioned response, where food access serves as the unconditioned stimulus and time of day as the temporal conditioned stimulus. Of course, this conditioning hypothesis requires the involvement of a circadian timing mechanism in the recognition of the time-of-day conditioned stimulus, since FARs are not seen when the period of the feeding schedule deviates too far from 24 hours. Second, there are motivational factors which may directly affect the overt expression of the FAR and thereby obscure the behavior of the underlying oscillator. For example, under ad lib conditions the expression of the FAR may be abolished due to the loss of food motivation which accompanies restoration of normal levels of food intake and body weight (in those studies which report food intakes, feeding schedules have been shown to reduce daily intake to about 75% of ad lib levels; e.g., [5]). In addition, under deprivation conditions, prolonged starvation may lead directly to non-specific decreases in activity levels. The purpose of the present experiments was to determine whether long-term persistence of FARs could be uncovered in the absence of a circadian feeding schedule. Whatever the mechanism of the damping of FARs under such conditions, it is clearly not possible to determine the long-term operating characteristics of the food-anticipatory oscillator from the limited persistence of the overt FAR which follows the cessation of the feeding schedule. The demonstration of longterm persistent FARs would strengthen the evidence for a self-sustaining, food-entrainable circadian oscillator in the rat, and permit the analysis of its free-running behavior as well as potential coupling relationships between the two circadian systems (photic and food-entrainable).
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Subjects and Apparatus Eighteen male albino Sprague-Dawley rats (Charles River), weighing approximately 300 g at the start of the experiments, were maintained in modified Wahmann running wheels. The side cage of each wheel was removed and replaced by an automated rotary feeding device which moved powdered rat chow in and out of the reach of the animal. The animal was therefore confined to the wheel compartment. The feeding device consisted of 4 food cups, each of which held more than 2 day's diet. When a feeding schedule was in effect, a fresh food cup was presented to the animal for 2 hours each day. Under ad lib, the (partially depleted) food cup was replaced by a fresh cup at the same time each day, thus providing continuous access to fresh food. Water was freely available throughout the experiments from a standard bottle mounted on the wall near the feeder• The running wheels were housed three to a cabinet, one per shelf. A single 25 W incandescent light was mounted on each shelf of the cabinet, and each cabinet had an independently programmable lighting schedule. Each revolution of the wheel was recorded on an Esterline-Angus event recorder• These records were cut into 24 hour strips and then pasted on boards with each day's record below the previous day, to construct standard circadian actograms. The cabinets were entered about once per week to replenish food and water supplies and to provide routine maintainance. This was done under dim red light if the cabinet was scheduled for darkness at that time.
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FIG. 1. Wheel running record from a representative group 1 animal. Successive days are plotted from top to bottom on the y-axis and time of day (24-hour span) on the x-axis. The animal was maintained throughout the experiment under a light-dark cycle with the dark segment indicated by the black bar above the record. During the period marked "'A," a restricted feeding schedule was in effect which allowed two hours of food access each day beginning at the vertical line in the record. During the period marked "B," the animal was exposed to complete food deprivation. At all other times, free access to food was allowed.
Procedures In an earlier study, Bolles and Moot [2] noted that an FAR which had damped out during ad lib feeding could be rapidly reinstated by reimposition of food deprivation; however, they did not formally present the results of this procedure. This observation suggested the use of the " m e m o r y " paradigm described below as a means of uncovering persisting rhythmicity of the food-entrainable oscillator after the complete damping of the overt FAR. During an initial baseline period of at least 2 weeks (Phase 1), the animals were permitted ad lib access to food while they acclimated to the wheels and the operation of the feeder device. In Phase 2, the animals were maintained under a 24 hr feeding schedule for at least 20 days. The ad lib baseline and the feeding schedule were both conducted under LD 14:10 (lights on at 2100 hr), and the feeding schedule provided 2 hrs of food access each day near the middle of the
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light period (0400-0600 hr). In Phase 3, the animals were returned to ad lib feeding; on the last day of the feeding schedule, the food became available at the usual time and then remained available continuously thereafter. During the second ad lib period, six animals remained under the same LD cycle (group 1), six were subjected to a 10 hr delay phase-shift of the LD cycle on the first day of ad lib (group 2), and six were maintained under constant light (LL) beginning on the first ad lib day (group 3). At various intervals after the beginning of Phase 3 (7 to 50 days), the animals in all groups were completely food deprived for four to seven days, and then returned to ad lib. The periods of deprivation began at different times of day for the different animals in order to rule out the possibility that running activity was triggered by the time of food removal. In addition, some animals in group 3 were subjected to repeated periods of deprivation, with each period separated from the others by at least three days of intervening ad lib feeding.
R E S U L T S
Representative results from one animal in each group are shown in the figures (Fig. 1, group 1; Fig. 2, group 2; Fig. 3, group 3). The results from all animals were quite similar and will be described together. As is typical of the nocturnal rat under LD and ad lib feeding conditions, wheel running in Phase 1 was mainly confined to the dark segment of the LD cycle. When the food access schedule was imposed (Phase 2), all animals developed FARs: robust wheel running occurred in the hours preceding each daily feeding. These FARs developed gradually over the 3-7 days after the introduction of the feeding schedule. In addition to the occurrence of FARs, the photic (nocturnal) wheel running rhythm sometimes showed changes during Phase 2; these included reduced magnitude and/or transient phase disruptions (usually advances). In other animals, the nocturnal activity rhythm was unaffected
ROSENWASSER, PELCHAI'
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by the feeding schedule. Despite these differences, all animals showed two daily components of running activity during Phase 2, one during the dark and one in anticipation of feeding. At the beginning of Phase 3 (return to ad lib), the FARs damped quickly; the animals showed 0-2 days of persisting anticipatory activity. In addition, the photic rhythms responded in typical fashion to the different manipulations of the lighting schedule in the different groups: animals in group 1 continued to show their normal nocturnal running rhythm,
animals in group 2 phase-shifted to the new LD phase in about one week, and animals in group 3 showed free-running activity rhythms with greater-than-24 hr periods under LL. The clarity and persistence of free-running rhythms in group 3 varied considerably, which is typical of rat activity rhythms under LL. The periods of deprivation imposed during Phase 3 occurred many days after the complete damping of overt foodanticipatory activity. Nevertheless, all animals in group ! showed robust running activity at the time of the previously
C I R C A D I A N MEMORY
29
scheduled daily feeding. This " m e m o r y " for feeding time could be obtained as many as 50 days after the termination of the original feeding schedule and the beginning of ad lib feeding; no longer intervals were tested. Similarly, animals in groups 2 an 3 showed robust deprivation-induced running at the time that feeding would have occurred under the previous feeding schedule, relative to the now shifted (group 2) or free-running (group 3) photic rhythm. In other words, all animals in all groups showed deprivation-induced running which began about 8--9 hours before the onset of the photic activity rhythm, the same phase relation which prevailed between the two rhythms under the original feeding schedule (we would expect the running induced during the deprivation tests to be approximately centered at the extrapolated time of food onset, as is seen during deprivation tests which immediately follow the termination of a feeding schedule; cf., [5]). In some animals, the deprivation-induced activity developed over 2-3 days, while in others, it seemed to persist for 2-3 days after the end of the deprivation test and the return to ad lib. In general, it appeared that deprivation-induced activity developed more slowly and was abolished more quickly with relatively longer intervening ad lib periods. However, all animals showed the induced activity at all tested intervals. The results with repeated memory tests in group 3 animals were more variable: In some animals, the occurrence of deprivation-induced activity became less robust with each test, and/or could not be elicited during the third or later tests. The animal shown in Fig. 3 showed a bout of activity at the appropriate phase relative to the free-running photic activity rhythm during the first two memory tests, but the deprivation-induced increase in activity seemed to occur too early in the circadian cycle during the third memory test, 75 days after the termination of the original feeding schedule. It is possible that this result was due to the extreme damping of the photic free-running rhythm seen by this point in the record. Thus the animals in all groups appeared to " l e a r n " the circadian phase of food availability during the feeding schedule, to " r e m e m b e r " the phase of food access over many days of intervening ad lib feeding, and to " e x p e c t " to be fed at the appropriate time, relative to the photic rhythm, when subsequently subjected to complete deprivation. DISCUSSION
In this study, rats demonstrated an ability to remember the relative circadian phase (of the photic rhythm) at which feeding occurred under a previous daily food access schedule. These results are in good agreement with those of two recent studies which appeared while this report was in preparation. Coleman et al. [6] have presented results from a paradigm very similar to our group 3, and also report that the photic and food-entrainable oscillators seem to free-run in parallel under ad lib access to food. In addition, they also presented one case in which dissociation between the two rhythms occurred (see Fig. 3 in the present report). The conditions under which such dissociations occur cannot be specified at present. In addition, Mori et al. [15] have recently shown that persisting food-related drinking rhythms may free-run in parallel with the photic drinking rhythm under ad lib feeding conditions. While it may be possible to explain the present results with reference to only a single light-entrainable oscillator, previous studies [5, 6, 7, 8, 18, 22, 25, 26] strongly support
the idea that F A R ' s depend on a separate, food-entrainable circadian oscillator. Thus we suggest that the ability to remember the relative phase of the feeding schedule, as shown in the present study, may require the involvement of both a food-entrainable circadian oscillator as well as the classical, photically entrainable circadian oscillator. In this scheme, the phase of the food-entrainable oscillator is controlled by the feeding schedule while it is in effect. However, during the subsequent ad lib conditions the food-entrainable oscillator continues to operate, but is controlled by (coupled to) the photic oscillator. On the other hand, overt anticipatory behavior is expressed only under conditions of relative or complete food deprivation or body weight reduction (i.e., during the feeding schedule, during the initial few days of ad lib feeding, and during the deprivation tests). This model of the rat's memory for feeding time requires that the coupling relationship between the food- and lightentrainable oscillators be dependent on environmental conditions, in particular, the presence or absence of a feeding schedule. This seems plausible, since it is well known that 24-hr food-anticipatory rhythms can exist concurrently with non-24-hr free-running photic rhythms under L L [5,9]. We therefore suggest that under a feeding schedule, the foodentrainable oscillator is entrained by the feeding schedule and is "'uncoupled" from the photic oscillator, while under ad lib feeding, the food-entrainable oscillator becomes coupled to and phase shifts or free-runs in parallel with the photic oscillator. Such a flexible coupling relationship between circadian oscillators may allow animals to synchronize their behavioral activity ("foraging") with both geophysically stable and predictable environmental periodicities (i.e., LD cycles) as well as unstable environmental periodicities (i.e., food availability). This coupled oscillator system may function as a "continuously consulted clock" (cf., [19]) in the adaptive temporal coordination of behavior. (It seems most likely that the SCN is a critical anatomical locus for the photic oscillator, while recent work suggests that the ventromedial nucleus (VMH) may be critical for the expression of FARs [ 10,11]. Neural connections which could potentially mediate coupling between these two nuclei have been demonstrated both anatomically and electrophysiologically [16,23].) Our hypothesis seems to further require that the two oscillators be capable of stable coupling at a n y mutual phase relationship during ad lib; a rigorous test of this hypothesis would require replicating this study using various different food access times during the feeding schedule, since in the present study all animals were " t r a i n e d " with the same phase relationship between the food access and light-dark schedules. It is also interesting to note that the phase of the food-entrainable oscillator appears not to be influenced by the spontaneous feeding rhythm which occurs in phase with the photic activity rhythm under ad lib conditions. Of course, the spontaneous feeding rhythm is of much lower amplitude than the " f o r c e d " feeding rhythm which occurs under a feeding schedule, and may simply be too weak a stimulus to entrain the food-entrainable oscillator. Additional evidence is available to further suggest that the two oscillators may interact through " w e a k " coupling mechanisms. Changes in the phase (under LD) or period (under LL) of the photic activity rhythm have been seen following the introduction and removal of a feeding schedule [5, 7, 9], and " b e a t " phenomena have been reported to occur under L L when the two rhythms periodically overlap [5]. Furthermore, a recent report [24] has suggested that the strength of interaction between the two concurrently ob-
3(I
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s e r v e d r h y t h m s d e p e n d s upon the similarity of t h e i r periods o r upon their mutual p h a s e relationship, as predicted by oscillator theory (cf., " M a g n e t E f f e c t s " I28]; see also 117] for an a l t e r n a t i v e model which m a k e s similar p r e d i c t i o n s a b o u t coupling strength). T h e s e results d e m o n s t r a t e the effects of the feeding schedule, or the f o o d - e n t r a i n a b l e oscillator, on the photic activity r h y t h m . H o w e v e r , in the present report we d e m o n s t r a t e that the photic s y s t e m m a y influence the b e h a v i o r of the F A R , at least during ad lib feeding c o n d i t i o n s w h e n the two s y s t e m s seem to p h a s e shift or free-run in parallel. T h e r e f o r e , we t e n t a t i v e l y suggest that a mutual coupling relationship exists b e t w e e n t h e s e two oscillator systems. A n o t h e r r e c e n t s t u d y s u p p o r t s the h y p o t h e s i s that the f o o d - a n t i c i p a t o r y oscillator m a y be c a p a b l e of l o n g - t e r m self-sustained r h y t h m s . S t e p h a n [22] has s h o w n a p p a r e n t f r e e - r u n n i n g F A R s in S C N - l e s i o n e d a n i m a l s m a i n t a i n e d on feeding s c h e d u l e s j u s t outside the circadian range of e n t r a i n m e n t . While S t e p h a n ' s data m a y show persisting
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F A R s in the p r e s e n c e of a Cnon-entraining) food s c h e d u l e and in the a b s e n c e of the photic oscillator, our m e m o r y paradigm d e m o n s t r a t e s persisting F A R s in the p r e s e n c e o f the photic oscillator but in the a b s e n c e of a feeding schedule, It would t h e r e f o r e be of interest to repeat the m e m o r y experi m e n t s in S C N - l e s i o n e d animals, in an a t t e m p t to e x a m i n e the " t r u e " free-running characteristics o f the FAR oscillator. In s u m m a r y , the results of the present sludy, when considered along with previous work, support the following c o n c l u s i o n s : t l) Feeding cycles e n t r a i n a circadian oscillator w h i c h c o n t i n u e s to o p e r a t e after the t e r m i n a t i o n o f the feeding s c h e d u l e , regardless o f i n t e r v e n i n g ad lib feeding conditions: (2) U n d e r ad lib, this oscillator free-runs or p h a s e shifts in parallel with the photic oscillator, i.e., they are c o u p l e d ; ~3) T h e b e h a v i o r a l e x p r e s s i o n of the lboda n t i c i p a t o r y oscillator requires c o n d i t i o n s of at least rehttive food d e p r i v a t i o n (or body weight loss): i4) T h e p h a s e of the f o o d - a n t i c i p a t o r y oscillator is not influenced by the spontaneous feeding r h y t h m which o c c u r s u n d e r ad lib conditions.
REFERENCES
1. Bolles, R. C. and J. DeLorge. The rat's adjustment to a-diurnal feeding cycles. J Comp Physiol Psychol. 55: 760-762. 1962. 2. Bolles, R. C. and S. A. Moot. The rat's anticipation of two meals a day. J Comp Physiol Psychol 83: 510-514, 1973. 3. Bolles, R. C. and L. W. Stokes. Rat's anticipation of diurnal and a-diurnal feeding. J Comp Physiol Psychol 69: 290-294, 1965. 4. Boulos, Z. and M. Terman. Food availability and daily biological rhythms. Neuros'ei Biobehav Rev 4:11%131, 1980. 5. Boulos, Z., A. M. Rosenwasser and M. Terman. Feeding schedules and the circadian organization of behavior in the rat. Behav Brain Res I: 39-65, 1980. 6. Coleman, G. J., S. Harper, J. D. Clarke and S. Armstrong. Evidence for a separate meal-associated oscillator in the rat. Physiol Behav 29:107-115, 1982. 7. Edmonds, S. C. and N. T. Adler. Food and light as entrainers of circadian running activity in the rat. Physiol Behav 18: 915919, 1977. 8. Edmonds, S. C. and N. T. Adler. The multiplicity of biological oscillators in the control of circadian running activity in the rat. Physiol Behav 18: 921-930, 1977. 9. Gibbs, F. P. Fixed interval feeding does not entrain the circadian pacemaker in blind rats. Am J Physiol 236: R249-R253, 1979. 10. Inouye, S.-I. T. Ventromedial hypothalamic lesions eliminate anticipatory activities of restricted daily feeding schedules in the rat. Brain Res 250: 183-187, 1982. 11. Krieger, D. T. Ventromedial hypothalamic lesions abolish food-shifted circadian adrenal and temperature rhythmicity. Endocrinology 106: 64%654, 1980. 12. Krieger, D. T. and H. Hauser. Comparison of synchronization of circadian corticosteroid rhythms by photoperiod and food. Proc Natl Acad Sci USA 75: 1577-1581, 1978. 13. Krieger, D. T., H. Hauser and L. C. Krey. Suprachiasmatic nuclear lesions do not abolish food-shifted circadian adrenal and temperature rhythmicity. Science 197: 398-399, 1977. 14. Moore-Ede, M. C., F. M. Sulzman and C. A. Fuller. The Clocks that Time Us. Cambridge, MA: Harvard Press, 1982. 15. Mori, T., K. Nagai and H. Nakagawa. Dependence of memory of meal time upon circadian biological clock in rats. Physiol Behav 30: 259-265, 1983.
16. Oomura, Y., T. ()no. H. Nishino. H. Kita, N. Shimizu, S. ishizuka and K. Sasaki. Hypothalamic control of feeding behavior: Modulation by the suprachiasmatic nucleus. In: Biological Rhythms and their Central Mechanism, edited by M. Suda, O. Hayaishi and H. Nakagawa. Amsterdam: Elsevier/North Holland, 1979, pp. 295-308. 17. Pavlidis, T. Spatial and temporal organization of populations of interacting oscillators. In: The Molecular BasL~ o f Circadian Rhythms. edited by J. W. Hastings and H. G. Schweiger. Berlin: Dahlem Konferenzen, 1976, pp. 131-148. 18. Phillips, J. L. M. and P. J. Mikulka. Effects of restricted ff)od access upon locomotor activity in rats with suprachiasmatic nucleus lesions. Phvsiol Behav 23: 257-262, 1979. 19. Pittendrigh, C. S. Perspectives in the study of bioh)gical clocks. In: Perspectives in Marine Biology, edited by A. A. BuzzatiTraverso. Berkeley: University of California Press, 1958. pp. 239-268. 20. Richter, C. P. A behavioristic study of the activity of the rat. Comp Psyehol Monogr 1: 1-55, 1922. 21. Rusak, B. and I. Zucker, Neural regulation of circadian rhythms. Physiol Rev 59: 449-526, 1979. 22. Stephan, F. K. Limits of entrainment to periodic feeding in rats with suprachiasmatic lesions..I (otnp Phvsiol 143: 401-410, 1981. 23. Stephan, F. K., K. Berkley and R. Moss. Efferent connections of the rat suprachiasmatic nucleus. Neuroscience 6: 2625-2641, 1981. 24. Stephan, F. K., J. A. Donaldson and A. Robbins. Food and light as entrainers of circadian rhythms. Soc Neuro~ci Abstr 7: 857, 1981. 25. Stephan, F. K., J. M. Swarm and C. L, Sisk. Anticipation of 24-hour feeding schedules in rats with lesions of the suprachiasmatic nucleus. Behav Neural Biol 25: 346-363, 1979. 26. Stephan, F. K., J. M. Swarm and C. L, Sisk. Entrainment of circadian rhythms by feeding schedules in rats with suprachiasmatic lesions. Behav Neural Biol 25: 545-554, 1979. 27. Sulzman, F. M., C. A. Fuller and M. C. Moore-Ede. Comparison of synchronization of primate circadian rhythms by light and food. Am J Physiol 243: R130-R135, 1978. 28. Von Holst, E. Die relative koordination als phanomen und als methods zentralnervoser funktionsanalyse. Ergeb Physiol Biol Chem 43: 228, 1939. Translated in The Behavioral Physiology qf Animals and Man, vol l, translated by R. Martin. Coral Gables: University Miami Press, 1973, pp. 33-135.