Food deprivation and reinstatement phase shifts rat activity rhythms in constant light but not constant dark

Physiology & Behavior, Vol. 50, pp. 167-171. ©PergamonPress plc, 1991. Printedin the U.S.A.

0031-9384/91 $3.00 + .00

Food Deprivation and Reinstatement Phase Shifts Rat Activity Rhythms in Constant Light But Not Constant Dark G R A H A M E J. C O L E M A N A N D A N D R E W J. P. F R A N C I S D e p a r t m e n t o f Psychology, La Trobe University, Bundoora, Victoria, Australia, 3083

Received 23 M a y 1990 COLEMAN, G. J. AND A. J. P. FRANCIS. Food deprivation and reinstatement phase shifts rat activity rhythms in constant light but not constant dark. PHYSIOL BEHAV 50(1) 167-171, 1991.--Three-day periods of food deprivation followed by reinstatement have produced phase shifts in the activity rhythms of the Australian marsupial, Sminthopsis macroura. However, no clear effects have been shown in rats. In the f'LrStof two experiments, rats in constant dark (DD) failed to show such phase shifts but when constant light (LL) was introduced it was possible to induce phase shifts. Because there was considerable disruption of activity rhythms, a second experiment was conducted using lower light levels which demonstrated clear phase shifts that were mainly phase delays late in the rats' inactive periods and advances late in the active periods. The size of the phase shifts was highly correlated with wheel-running activity levels and free-running period but not with proportional changes in activity associated with the food deprivation schedule: The results indicate that either the effects of food deprivation/reinstatement are augmented by LL or that the increased free-running period induced by LL produces larger phase shifts. Because no phase shifts at all were observed under DD, the former interpretation is preferred. Circadian rhythms

Phase shifts

Food deprivation

IT has been reported that 3-day periods of food deprivation in constant dark (DD) produce phase shifts in the free-running activity rhythms of the Australian marsupial Sminthopsis macroura (5). Although it was not previously reported, there is also evidence for deprivation-induced phase shifts in activity of rats in DD (2,4) and, more recently, under light/dark (LD) cycles (3). There are three aspects of the food deprivation and reinstatement (Fd/r) procedure which could affect an animal: a) the circadian time of food removal, b) the circadian time of food reinstatement and c) the time interval between a and b. The phase shifts observed in S. macroura appeared to occur on the day on which food was reinstated (5). If this is the case, then it suggests that food may act as a stimulus to produce these phase shifts. Such an effect would be behaviorally similar to that observed following pulses of light administered to animals (6, 7, 12) and man [e.g., (8)] and of exposure to a running wheel (13) and to a novel cage or to a conspecific (11) in animals. It is also possible that either the time of food removal or duration of deprivation is the relevant factor inducing phase shifts. The actual duration of food deprivation (and therefore, the intensity of its effects) depends both on the circadian time at which food was removed and on the ingestive behavior of the rat immediately prior to the removal of the food. With respect to the circadian time of deprivation, it could be argued that a rat would be hungrier when deprived during its active (and feeding) period than when deprived during its rest phase. If this is the case, then to the extent that duration of deprivation may be functionally associated with Fd/r-induced phase shifts, rats should show a phase

response to circadian time of food deprivation and/or reinstatement. It is difficult to make predictions about the rhythmic behavioural consequences of duration of food deprivation because the relatively slow changes in relevant gut and metabolic processes make it hard to define the onset time of a deprivation signal. Food replenishment following deprivation represents a more definite signal because, even during the photophase of an LD cycle, hungry rats will eat as soon as food is available. Therefore, for the practical reason the food replenishment following deprivation is a reasonably discrete stimulus, and because S. macroura appeared to phase shift following food reinstatement, there is a case for studying the role of food reinstatement in activity phase shifts in rats. So observations of phase shifts in activity rhythms upon presentation of food following a period of deprivation suggest that some aspect of food, or its absence, is acting as a zeitgeber. This is consistent with the widely replicated observation that a dally meal presented at the same time each day can entrain activity rhythms in rats [e.g., (1, 2, 4, 14)]. Given, however, that there is evidence for separate meal-associated and light-associated oscillators in rats (4), the question of whether meal feeding schedules and Fd/r schedules influence activity rhythms via similar mechanisms remains to be determined. Nevertheless, if Fd/r acts similarly to other zeitgebers, it should be possible to construct a phase response curve (PRC) for the amount and direction of phase shifts induced by meals presented at varying circadian times. Such PRCs have been constructed for light

167

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COLEMAN AND FRANCIS

(6,12) and exposure to a conspecific (11) in a variety of species. One difficulty with this proposal is that phase shifts in response to food reinstatement have not been reported in rats, despite a number of published studies which have used this methodology (2,4). In fact, a close inspection of the activity records published in these studies suggests that some phase shifts may have occurred, but it is difficult to be sure because only short intervals of free-running activity were recorded between food deprivation episodes. If the phenomenon is to have any generality then it must be clearly demonstrated in species other than S. macroura. The aim in the research reported here was to establish the conditions under which food reinstatement could induce phase shifts in wheel-running activity rhythms in rats. METHOD

Animals Twenty male, Long-Evans rats, 88 and 91 days old at the commencement of the experiment, were used in Experiments 1 and 2 respectively.

Apparatus Rats were housed in individual cages in a sound-attenuated, air-conditioned laboratory with an ambient temperature of 2 1 - 2 ° C . Cages measuring 20 x 21 × 30 cm were installed in a steel-framed rack. Drinking nipples were placed at the rear of each cage and a running wheel with a wire mesh floor and clear perspex face was attached to the front of the cage. Wheel-running activity was sampled at 15-min intervals by a data acquisition computer that was remote from the laboratory. The laboratory was entered at irregular intervals for maintenance and, during the period of ad lib feeding, for food replenishment. Food consisted of GR2 + (Clark-King, Australia) rat pellets which were crushed into a dry mash and placed in glass containers inside the cage.

Procedure In Experiment 1, rats were placed on a 12:12 LD cycle (lights on at 0600) and ad lib food for 16 days. Following this, DD was introduced and, 17 days later, rats were deprived of food at 1800 hours and the laboratories were cleaned. Three days later, food was reinstated at circadian times (CT) of 0400, 0800, 1200, 1600, 2000 and 2400 h with 3 or 4 rats fed at each time. Then followed 15 days of ad lib food availability and another 3-day deprivation period with food removed at 1700 h and reinstated at the same CT's as previously but, for each rat, at a time different from that used previously. Thus each rat had food reinstated in DD at two different CT's. Fifteen days later rats were placed in constant light (LL) (30---40 lux) at 1800 h and after ten days in LL were deprived of food for 3 days with food removed at 1445 h and reinstated at CT 1200 estimated from the predeprivation free-running rhythm. After a further 18 days, the experiment was terminated. In Experiment 2, rats were placed on a 12:12 LD cycle (lights on at 0600) and ad lib food for 15 days. Following this, low intensity LL was introduced (approximately 10 lux) for 24 days at which time a 3-day deprivation period was introduced at 1430 h. Food was reinstated also at 1430 h and rats continued to free run for a further 13 days after which the experiment was terminated. In both experiments, the free-running period of the wheel-

running rhythm was calculated by eye-fitting lines through activity onsets for at least 10 days of stable rhythmicity before the introduction of food deprivation and 10 days after food reinstatement. The amount and direction of phase shift was calculated for the day of food reinstatement by extrapolating the line fitted to onsets prior to food deprivation forward and, if necessary, the line fitted through activity onsets following food deprivation was extrapolated backwards. In Experiment 1, food reinstatement times were varied across the circadian day to observe the possible effects of the time of food reinstatement on phase shifts. In Experiment 2, food reinstatement was restricted to the same actual time of 1430 h. RESULTS

Experiment 1 All rats free ran with a period greater than 24 h when DD was introduced (Fig. 1). When the deprivation periods were introduced during DD, no rats showed any evidence of phase shifts. When deprivation was introduced during LL, however, a number of rats showed a clear phase shift in the free-running period. Several rats also showed substantial disruption of freerunning activity which appeared to become more severe following food deprivation (Fig. 2). In all rats there was some indication that they were not stably entrained to the LD cycle at the time DD was introduced (Figs. 1 and 2) indicating that perhaps they had not become properly acclimated to the laboratory after removal from the animal house where they had been held prior to the experiment.

Experiment 2 Following the introduction of LL, all rats free ran with a period greater than 24 h (mean free-running period=24.73 h). Following the 3-day deprivation period, clear phase shifts were observed in the free-running activity rhythms of 14 out of 20 rats. Of the rats which showed phase shifts, 5 showed delays (Fig. 3) while 9 rats showed phase advances (Fig. 4). At the lower light levels used in this experiment, there was less disruption of free running activity rhythms than was observed in Experiment 1. Although food was always reinstated at 1430 h, the corresponding CT's varied and were classified into 4 h blocks beginning at 0000 h. One rat with a CT in the 0000 h block showed a phase advance of 1.9 h. One rat in the 0400 h block showed a phase delay of 1.2 h while 2 rats in the 0800 h block showed delays of 5.2 h and 7 h. In the 1200 h block, 2 rats showed delays of 5.8 and 0.6 h, while one rat showed a 5.4 h advance. In the 1600 h block, the mean phase shift was an advance of 2.9 h with 6 rats advancing, and one delaying. In sum, although there was insufficient variation in CT's to permit the construction of a phase response curve, rats tended to show phase delays late in their inactive period, and phase advances late in their active period. There was a clear difference in the characteristics of the rhythms in the two rats depicted in Figs. 3 and 4. Rat #15 (Fig. 3) showed a unimodal free-running rhythm with a clear, welldef'med onset whereas rat #5 (Fig. 4) showed a bimodal rhythm with onsets less clearly defined. This variability amongst the rats was similar to but much less than that observed under the higher light levels used in Experiment 1. No systematic relationship between activity patterns and magnitude or direction of phase shifts was observed. Running-wheel revolutions recorded for the circadian day prior to the removal of food ranged from 55 to 4226, with a mean of 1042.36. On the last circadian day on which rats were

FOOD DEPRIVATION AND PHASE SHIFTS

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deprived, the range of running-wheel revolutions was from 313 to 4801 with a mean of 1928.81. In order to investigate the relationships between free-running period, phase shifts, activity levels before and on the last day of food deprivation and the ratio of these two activity levels, Pearson product moment correlations were calculated. From Table 1 it is clear that activity levels correlate highly with both free-running period and the size of the phase shift but not with the proportional increase in activity (i.e., the ratio of activity on the last day of deprivation divided by the activity on the day before deprivation). DISCUSSION These experiments clearly demonstrate that periods of food deprivation can induce phase shifts in free-running activity rhythms of rats, but this effect appears to be restricted to LL. In this respect, rats appear to be different from the marsupial S. macroura (5). It appears that light may exert some direct effect on the rat circadian system to augment the effects of depriva-

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FIG. 4. Double plot of wheel-running activity of animal #5 from Experiment 2. Conventions are the same as in Fig. 3.

delays and long periods, while rats with lower activity levels tended to show phase advances and shorter periods. Certainly the results reported here do not support the view that the relative increase in wheel running affects the size of phase shifts in rats. It may be that more active rats are more reactive to the environment and this is reflected in larger increases in wheel-running during food deprivation and a greater response to food reinstatement than are less active rats. Thus it may not be the mere "all or nothing" access to a running wheel which produces the effects reported in the earlier studies and it is essential that the physiological events associated with such access be studied further. One hormone associated with changes in activity levels in rats is corticosterone. It has been widely reported that, in rats, a peak in plasma corticosterone occurs in anticipation of a daily meal. Changes in glucocorticoid levels might be involved in the observed phase shifts, although this is not to imply that the mechanisms underlying the phase shifts are the same as those involved in meal anticipation. However, the magnitude of the peak in plasma corticosterone, which occurs in anticipation of a daily meal, is not different under DD or LL conditions (10). It therefore appears that the interaction between lighting conditions and the effects of food deprivation/reinstatement may not be related to changes in glucocorticoids. Indirect evidence for this assertion comes from the report that although ACTH responses in rats differ under LL compared to DD, adrenocortical response is not different (8). Although an attempt was made in this study to systematically vary the circadian time of day at which food was reinstated following deprivation in DD, this was not done in Experiment 2 where the aim was simply to demonstrate whether or not phase shifts could be observed in LL. However, from the limited distribution of CT's for food reinstatement actually used, it appeared that rats tended to phase delay late in their inactive period, and to advance late in their active period. This is similar to the phase responses of a variety of species to light pulses (6), but different from the PRC for social entrainment in hamsters (11). It remains to be determined how robust this observation is and whether it implies a common underlying mechanism with the effects of light. It is not entirely clear whether phase shifts were associated with food reinstatement or whether they occurred at an earlier time. In Fig. 4, for example, the phase shift appeared to occur after food reinstatement. In Fig. 3, on the other hand, activity onset may have phase shifted prior to food reinstatement. It remains for this issue to be systematically investigated. However, if food reinstatement is to act as a zeitgeber, it should be possible to construct a comprehensive PRC for food reinstatement in both rats (in LL) and S. m ac r our a (in DD).

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