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Restricted feeding facilitates time–place learning in adult rats
Behavioural Brain Research 134 (2002) 283 /290 www.elsevier.com/locate/bbr
Research report
Restricted feeding facilitates time place learning in adult rats /
Nikolai V. Lukoyanov *, Pedro A. Pereira, Rui M. Mesquita, Jose´ P. Andrade Department of Anatomy, Porto Medical School, Alameda Prof. Hernaˆni Monteiro, 4200-319 Porto, Portugal Received 2 January 2002; received in revised form 6 February 2002; accepted 6 February 2002
Abstract Many species can acquire time-of-day discrimination when tested in food reinforced place learning tasks. It is believed that this type of learning is dependent upon the ability of animals to consult their internal circadian pacemakers entrained by various environmental zeitgebers, such as light /dark cycles and scheduled restricted feeding. In the present study, we examined, (1) whether rats can acquire time-of-day discrimination in an aversively motivated water maze task wherein an escape platform is located in one position in the morning and in another position in the afternoon; (2) whether time-of-day cues provided by the light- and feedingentrainable pacemakers may have divergent impacts upon the ability of rats to learn this task. Two groups of rats, both maintained on 12-h light:12-h dark cycle, were used; in one group, animals had free access to food, whereas in the other, they were subjected to a restricted feeding protocol (60% of food consumed by rats fed ad libitum, once daily). Despite the heightened difficulty of the task, food-restricted rats were apparently able to acquire associations between two different platform positions and two different times of day, as indicated by the fact that the percentage of discrimination errors in this group declined progressively, as a function of training, and stabilized at the level of 229/9%. In contrast, rats that were fed ad libitum, even after extensive training, failed to perform the task above level of chance. These data indicate that time /place learning is a universal, reward-nonspecific, cognitive phenomenon. They furthermore suggest that the ability of animals to integrate spatial and temporal information can be dependent on the access to timing stimuli provided by the feeding-entrainable circadian system. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Time /place learning; Timed hypocaloric diet; Two-choice water maze; Win-stay strategy; Win-shift strategy; Corticosterone; Circadian rhythms
1. Introduction Substantial evidence from behavioral studies indicates that animals are capable of organizing their daily activities, particularly those devoted to feeding, by using time of day as a discriminative temporal cue. For example, the ability to associate different food locations with different times of day has been described in ants [39], fish [36], birds [1], and in mice housed in seminaturalistic environment [14]. Laboratory studies have yielded similar results. It has been reported that rats can learn to press one lever for food in the morning and the other in the afternoon [4,7]. It was also found that rats can learn a time-of-day discrimination T-maze task in which they are required to search for food in one arm of
* Corresponding author. Tel.: 351-22-509-6808; fax: 351-22550-5640 E-mail address: [email protected] (N.V. Lukoyanov).
the maze during a morning training session and in the other during an afternoon training session [25,26,29]. However, it was also reported that rats learn more readily to initiate food-seeking behavior at the appropriate time than to associate availability of food at specific locations with times of day and that relatively small proportion of them (approximately 60%) are able to acquire a criterion of 90% correct choices in a true time/place learning task [25,26]. Although it seems obvious that time /place learning is based upon the ability of animals to integrate spatial information with temporal cues, both the nature and the origin of these temporal cues remain enigmatic. One possibility is that animals can consult their circadian pacemaker entrained by light /dark cycles and known to be located in the suprachiasmatic nucleus of the hypothalamus [21,43]. However, the observations that time-of-day discrimination is not disrupted by complete ablation of this nucleus and by housing animals in
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constant light argues against this hypothesis [28,29]. It has also been suggested that time-of-day cues are provided by a circadian pacemaker entrained by timed restricted feeding, the anatomical location of which is presently unknown [10 /12,28,29,40]. Thus, it can be hypothesized that maintenance of animals on ad libitum feeding regimen would result in a loss of their ability to learn time-of-day discrimination tasks. This possibility, however, has not yet been addressed. Indeed, all above cited laboratory studies on time /place learning were performed using appetitively motivated behavioral paradigms [4,7,8,25,26,29], a condition which does not permit the use of animals fed ad libitum. This problem can be circumvented by the employment of a nonappetitively motivated place learning task, such as the Morris water maze [30], which utilizes the ability of rodents to learn the position of a submerged escape platform in a large tank filled with opaque water. We introduced one additional variable to this task */time of day */by training rats to search for the platform in one position in the morning and in another position in the afternoon. In doing so, we sought to examine whether rats can consult their internal circadian pacemakers, entrained by either light /dark cycles alone [10,29] or in combination with timed restricted feeding [9,28], in order to associate different locations of the escape platform with different times of day.
2. Methods 2.1. Animals Sixteen male Wistar rats derived from the colony of the Gulbenkian Institute of Science (Oeiras, Portugal) were used in the present study. Animals were housed individually, and maintained under standard laboratory conditions (20 /22 8C, lights on from 07:00 to 19:00 h, free access to food and water). At 8 weeks of age, rats were randomly assigned to one of two groups with eight animals in each. Rats in the first group were fed ad libitum with a standard laboratory chow (Letica, Spain), supplemented with salts and vitamins (AL group), while rats in the other group were fed 60% of the amount of food consumed by AL animals (FR group [3]). These FR rats were fed once a day either at 08:00 or at 11:00 h, following a random schedule ensuring that the feeding procedure cannot be used by the animals as a discriminative cue. Both groups of rats were maintained on their respective feeding regimen until the end of the experiment. At 20 weeks of age, all rats were handled 5 min per day for 5 consecutive days and submitted to behavioral testing. During the period of handling, blood samples (300 ml) were collected from the dorsal vein of the tail at 08:00, 13:00, and 18:00 h. These samples were
used to analyze circadian fluctuations in the serum levels of corticosterone. The handling and care of the animals were conducted according to the European Communities Council guidelines in animal research (86/609/UE). 2.2. Serum corticosterone levels The blood samples were centrifuged, and the serum was separated and stored at /20 8C until the time of assay. Serum corticosterone levels were determined by radioimmunoassay using a kit from ICN Biomedicals. All samples were analyzed in a single run and the intraassay coefficient of variation was 5.2%. 2.3. Water maze apparatus The behavioral apparatus used in this study has been described in detail previously [3,23]. Briefly, it consisted of a black circular tank, 1.8 m in diameter and 0.5 m deep. The tank was located in a corner of a room containing extra-maze cues, i.e. three posters and desk with computer. The position of the pool and of the extra-maze cues was kept unchanged throughout the period of behavioral testing. The apparatus was filled with water at room temperature (219/1 8C) to a depth of approximately 35 cm. The water was made opaque by adding a nontoxic paint. The pool was divided, by imaginary lines, into four equal-size quadrants. A black circular escape platform, 10 cm in diameter, was located 2 cm below the surface of water. It was positioned in the center of one of the quadrants during all morning training sessions and in the center of the opposite quadrant during all afternoon sessions. The swim path was recorded by a computerized video-tracking system (EthoVision V1.90, Noldus, The Netherlands). 2.4. Procedure The procedure for the water maze testing was essentially as described previously [3,23] except that animals were required to learn the two different platform positions, one in the morning (beginning at 09:00 h) and the other in the afternoon (beginning at 18:00 h). For acquisition, rats were given one morning and one afternoon training sessions per day for 30 days. However, in order to reduce the possibility that the animals can perform the task using a simple session-to-session alternation strategy, one of each five consecutive sessions was randomly omitted. Therefore, a total of 25 morning and 25 afternoon training sessions was given. Each session consisted of one testing trial followed by three reinforcing trials. Each trial begun by placing the rat into the water facing the pool wall at one of four starting points that were used in a pseudorandom order so that each point was used once
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in each session. However, the first trials of each session were always given from one of two starting points equidistant to both platform positions. The starting points were the same for all animals. Each trial ended when the rat climbed onto platform. If the rats did not find the escape platform within 60 s, the experimenter guided them to the platform. After climbing onto platform, all rats were allowed to remain there for 15 s. The intertrial interval was 30 s. After the last trial of every session, the rats were towel dried and placed in a holding cage close to a heater for 15 min. The distances swum by rats to find the escape platform were analyzed. In addition, the numbers of discrimination errors, defined as swims through the area corresponding to the afternoon platform position during morning training and vice versa, were registered. Since on the second, third and fourth session trials animals could use the information gained on the anterior trials of that session, only the pathways derived from the first session trials (testing trials) were analyzed. One day after completion of the task, animals received a single 60-s probe trial in which the platform was removed from the pool. All rats were released from the same starting point equidistant to both former platform positions. The number of times the rats swum through the areas where the platform had been located in the morning and in the afternoon (platform crossings) and the percentage of time spent in the two training quadrants corresponding to the morning and afternoon platform locations were recorded. 2.5. Statistical analysis The serum corticosterone levels were analyzed using repeated measures ANOVA. The swim distances to find the platform in the morning and in the afternoon were averaged over blocks of five consecutive sessions each and analyzed using repeated measures ANOVA. The errors made in the discrimination between the two platform positions were averaged over blocks of ten sessions each (five consecutive morning plus five consecutive afternoon sessions) and analyzed using repeated measures ANOVA. The discrimination errors made by rats on the first sessions following the omission tests were averaged over consecutive blocks of one morning session and one evening session. The time-of-day discrimination errors made by rats during the last training sessions, separately in the morning and in the afternoon, were averaged over blocks of five last morning and five last afternoon sessions each and analyzed using two-way ANOVA. The measures derived from the probe trial were analyzed using one-way ANOVA. Newman /Keuls post hoc test was used where appropriate. The data are expressed as the mean9/ S.E.M. Differences were considered as significant at the P B/0.05 level.
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3. Results 3.1. Serum corticosterone levels Serum levels of corticosterone found in blood samples collected at 08:00, 13:00 and 18:00 h are shown in Table 1. In rats that were fed ad libitum, the corticosterone levels were lowest in the morning and reached their highest values late in the afternoon. In food-restricted rats the highest concentrations of the hormone were found in blood samples collected in the morning and in the afternoon, while they were lowest in samples collected at 13:00 h. Although there was no significant main effect of feeding regimen on the corticosterone levels, ANOVA revealed a significant effect of sampling time (F2,28 /5.53, P B/0.01) as well as a significant interaction between feeding regimen and sampling time (F2,28 /9.40, P B/0.001). Post hoc tests indicated that corticosterone levels in AL rats were significantly higher at 18:00 than at 08:00 and at 13:00 h (P B/0.01). Likewise, in FR animals, corticosterone levels were significantly higher in samples collected at 08:00 and at 18:00 h than in those collected at 13:00 h (P B/0.001 and P B/0.01, respectively). Finally, the levels of corticosterone measured at 08:00 h were significantly higher in FR rats than in AL rats (P B/0.001). No significant differences between FR and AL groups were found when comparing serum corticosterone concentrations in samples collected at 13:00 and 18:00 h. 3.2. Swimming distances Distances swum by rats to find the hidden platform in the morning and in the afternoon are presented in Fig. 1. The overall repeated-measures ANOVA showed that food-restricted rats and rats fed ad libitum progressively improved their ability to locate both platform positions over the entire course of training (F4,112 /91.12; P B/ 0.0001). Although there were no significant effects of feeding regimen and time of day on this measure, ANOVA detected a significant interaction between these two variables (F1,28 /9.54; P B/0.005). Pairwise comparisons revealed that, in the afternoon, rats from AL group swam shorter distances than did FR animals Table 1 Serum levels of corticosterone (ng/ml) in blood samples collected from rats fed ad libitum (AL) and food-restricted (FR) rats at 08:00, 13:00 and 18:00 h Groups
08:00 h
13:00 h
18:00 h
AL FR
108923 242933**
113916 110918
195915* 181918*
Values are presented as mean9S.E.M. *, P B 0.01 vs. 08:00 h in AL group and vs. 13:00 h in both groups. **, P B 0.001 vs. 08:00 h in AL group and vs. 13:00 h in both groups.
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Fig. 1. The distances (in centimeters) swum by rats fed ad libitum (AL) and by food-restricted (FR) rats to reach a hidden platform in the morning and in the afternoon for each block of five consecutive training sessions. Note that, at the beginning of the training, rats in AL group located the platform faster in the afternoon than in the morning. *, P B 0.05 vs. AL group in the morning and FR group in the afternoon.
(F1,14 /22.85; P B/0.001). Moreover, AL rats performed significantly better in the afternoon than in the morning (F1,14 /9.91; P B/0.01). Post hoc tests showed, however, that this effect was significant only at the beginning of acquisition (P B/0.05 for the first and second session blocks) and, after 10 days of training, had became undetectable (Fig. 1).
3.3. Discrimination errors The percentages of errors made by rats in first session trials, averaged over blocks of ten (five morning plus five afternoon) consecutive sessions each, are shown in Fig. 2. The overall repeated-measures ANOVA applied on these data revealed a significant effect of session blocks (F4,56 /5.26; P B/0.01) indicating that acquisition of the task was associated with a significant decrease in the percentage of errors made in initial trials. This decrease, however, was not uniform in FR and AL groups as indicated by a significant main effect of feeding regimen on the error level (F1,14 /9.59; P B/ 0.01). In fact, only animals in FR group significantly improved their performance over the course of training (F4,35 /3.65; P B/0.01), whereas the corresponding decrease in the percentage of errors made by AL rats did not reach the level of significance (F4,35 /1.92; P / 0.13). At the end of training, FR rats performed significantly above chance level (P B/0.05), making at most 25% of incorrect choices. In contrast, animals in AL group, even after extensive training, failed to perform above chance level, making significantly greater number of errors relative to FR rats (P B/0.05 for the session blocks 3 /5).
Fig. 2. The percentages of incorrect choices for each block of ten training sessions (five consecutive sessions given in the morning plus five consecutive sessions given in the afternoon). The percentages of errors made by rats on the first sessions following the omission tests, averaged over consecutive blocks of two sessions (one given in the morning and the other in the afternoon), are indicated by solid symbols. Note a progressive decrease in the number of errors made by food-restricted (FR) rats. In contrast, animals fed ad libitum (AL) failed to succeed in this task, performing at close to chance level throughout the entire course of training. *, P B 0.05 vs. AL group.
The percentages of discriminative errors made by rats on the first sessions following the omitted sessions, averaged over blocks of one morning and one evening session each, are shown in Fig. 2. Repeated-measures ANOVA failed to reveal any significant difference between the percentages of errors made by rats on these sessions and on the sessions that were not preceded by the omissions, indicating that the performance of rats in either group was not disrupted by the omission tests. The percentages of errors in initial trials made by rats in each group during the last five training sessions, separately in the morning and in the afternoon, are presented in Fig. 3. ANOVA applied on these data revealed a significant effect of feeding regimen (F1,14 /
Fig. 3. The percentages of incorrect choices made by rats in the morning and in the afternoon averaged over the last five training sessions. Note that FR animals had similar error scores in the morning and in the afternoon, whereas rats fed AL performed particularly poorly in the morning, making far fewer errors in the afternoon. *P B 0.01 vs. AL group in the afternoon and FR group both in the morning and in the afternoon.
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7.06; P B/0.01), a significant effect of time of day (F1,14 /9.63; P B/0.05) and a significant interaction between the two variables (F1,14 /13.69; P B/0.01). Inspection of these data indicated that AL rats performed particularly poorly in the morning (809/8% of incorrect responses), making far fewer errors in the afternoon (149/5%; P B/0.01 for the difference). Unlike AL rats, animals in FR group had similar error scores during the morning and afternoon training sessions (199/13 and 259/9%, respectively). In addition, post hoc
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tests indicated that, whereas FR rats significantly outperformed AL rats during the morning training sessions (P B/0.01), the percentages of errors made in the afternoon did not significantly differ between the groups. 3.4. Probe trial The results of the probe trial are shown in Fig. 4. In this test, rats from FR and AL groups spent an identical proportion of time swimming in either of the two training quadrants corresponding to the morning and afternoon positions of the platform. Likewise, the numbers of times the animals crossed the areas where the escape platform had been located during the morning and afternoon sessions (platform crossings) were highly similar in either group. ANOVA failed to detect any significant effect of feeding on these parameters.
4. Discussion
Fig. 4. Performance of rats fed AL and FR rats on the probe trial. (A) Path taken by a representative rat in AL group. Paths like this were taken by rats in both groups. The two positions where the platform had been located during training sessions given in the morning and in the afternoon are indicated. Also shown are (B) number of times the rats crossed the former platform positions (platform crossings) and (C) percentage of time spent swimming within the two training quadrants. No significant differences between groups were found on this test. Furthermore, in either group, no differences between the measures corresponding to the two different platform positions were detected.
One of the main findings of the present experiment is that rats that were subjected to timed food restriction were apparently able to learn the time-of-day discrimination version of the water escape task, as indicated by the progressive decrease in the number of incorrect choices made by these animals over the course of training. This observation fits nicely the results of several previous studies showing that food-restricted rats can learn to use temporal cues to press a correct lever for food [4] or to find food in a T-maze [25,26,29]. Importantly, current data extend the previously reported findings revealing the capacity of food-restricted rats to acquire time-of-day discrimination not only in the appetitively motivated tasks but also when they are required to learn spatial locations of escape routes. Considered together, these results thus suggest that time/place learning is a universal, reward-nonspecific, cognitive phenomenon. On the other hand, we have additionally found that time-of-day discrimination in the water maze is quite difficult for rats to learn. Indeed, the animals approached the asymptotic performance only after they were given as many as 200 trials (taking into account the reinforcing trials); even so, the level of correct choices did not exceed 80%. For comparison, learning of the classical Morris water maze, in which the escape platform is located in a constant position, usually requires 20 /30 training trials [3,23,30]. It has been previously reported that rats have difficulty acquiring time-of-day discrimination in appetitively motivated tasks [8,25,26]. Another principal finding of this study is that, unlike food-restricted rats, animals that were fed ad libitum failed to acquire the temporal discrimination component of the task. This finding is illustrated by the fact that,
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even after a very extensive training, the number of correct choices made by these rats in first session trials remained at chance level. Yet, the distances swum to find the platform at either position did not differ between groups, except during the initial phase of training. Furthermore, the measures derived from the probe trial, that is platform crossings and percentages of time spent in training quadrants, were identical in AL and FR groups, suggesting that animals in both groups had comparable spatial learning and memory abilities. These data indicate that the low percentage of correct choices shown by AL rats is not related to spatial learning deficits, but rather, reflects their inability to associate spatial information with temporal cues. The observation that rats in FR group were able to acquire time-of-day discrimination, while those in AL group were not, can imply that animals in these two groups had access to different sets of temporal cues, provided probably by different circadian oscillators. In support of this view, we found that, whereas in animals that were fed ad libitum the serum levels of corticosterone were elevated only late in the afternoon, in those that were maintained on restricted feeding regimen the corticosterone levels were in addition prominently increased in the morning. The fact that the two peaks of corticosterone release were coincident either with the onset of nocturnal activity (in both groups) or with the anticipation of feeding (in FR group) indicates that they were entrained, respectively, by light /dark cycles [13] and by the scheduled daily meal [18,32,42]. Thus, taking into account that only rats from FR group performed the task above chance level, it is possible to assume that the ability to acquire time-of-day discrimination is largely dependent upon the access to temporal information provided by the food-entrainable circadian oscillator [25,26,28,29]. When analyzing the numbers of incorrect choices made by rats during the last 5 days of training, we unexpectedly found that the animals in AL group were far more precise in the afternoon (14% of errors) than in the morning (80% of errors). In other words, these animals adopted a perseverative behavioral response, known also as a win-stay strategy [15], */always to inspect first the presence of the platform in its afternoon position*/and applied it for both daily sessions. In contrast, animals in FR group adopted a win-shift behavioral strategy required to correctly perform the two-choice water escape task, as indicated by the observation that these rats were able to use two different navigation responses corresponding to the two different platform positions. The finding that AL rats favored to use the afternoon platform position as an escape route of first choice can imply that, during training, they gained more spatial knowledge in the afternoon, probably, due to the high circulating levels of corticosterone known to facilitate spatial learning [22,31,38,41], than in
the morning, when the levels of corticosterone were relatively low. This is consistent with the observation that, at the beginning of training, these animals learned the afternoon position of the platform faster than its morning position. However, such an interpretation conflicts with our finding that, at the end of training, the amount of spatial information acquired by AL rats was the same for both platform positions, as indicated by the distances swum during the last training sessions as well as by the results of the probe trial. Thus, while perhaps accounting for the different rates of initial learning shown by AL rats in the morning and in the afternoon, this hypothesis does not explain why the animals consistently used the win-stay strategy throughout the entire period of training. Given these considerations, it seems more likely that the simple win-stay strategy employed by rats in AL group is associated with their inability to use time of day as a discriminative stimulus and, therefore, might have for them an adaptive value. It can also be suggested that the difference between the two groups in the performance on the time /place task is related to the beneficial effect of food restriction on the cognitive abilities of rats. This hypothesis is strongly supported by the evidence indicating that restricted feeding enhances learning and memory in aged cognitively impaired rodents [19,20,34] and improves behavioral outcome following different types of brain lesions [6,16]. Surprisingly, the beneficial effects of restricted feeding on cognitive processes in healthy adult rats have never been convincingly demonstrated, despite the fact that different types of learning and memory were tested using different behavioral paradigms, such as the radial maze [17], Morris water maze [3,24], and operant conditioning [37] and passive avoidance [3] tasks. Furthermore, it has been recently shown, in different laboratories [3,24], that restricted feeding does not alter the performance of adult rats on the variable platform position version of the Morris water maze, a task known to rely on the ability of rats to use both the win-stay and the win-shift strategies. Nonetheless, it remains possible that the enhanced time /place learning shown by food-restricted rats in our experiment is associated with beneficial effects of restricted feeding on some other types of behavioral processes, which were not properly assessed in the above-cited studies. Future comparative experiments on the effects of this feeding regimen on animal behavior will help to clarify this issue. Taking into account the current data as well as the findings of previous studies [4,5,25,26,29], it appears that access to temporal information provided by the food-entrainable circadian system is indeed essential for the ability of animals to acquire time /place associations. However, it remains unknown how this temporal information is coupled with the spatial one and which
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regions of the brain are involved in this process. There is now strong evidence that the hippocampal formation plays a critical role both in the processing of spatial information and in associative learning [33]. Therefore, it is theoretically possible that the integrative cognitive processes underlying time/place learning are also subserved by hippocampal neuronal circuits. Interestingly, we have recently found that maintenance of rats on a hypocaloric diet similar to that used in the present experiment produces a 15% increase in the total number of synapses in the molecular layer of the dentate gyrus [3]. Bearing in mind that the vast majority of hippocampal afferents terminate in the dentate gyrus [2], it can be speculated that the synaptic changes in this area are somehow related to the increased flow of temporal information arriving to the hippocampal formation from the food-entrainable circadian system. Admittedly, although the idea that the increase in the number of synapses in the dentate gyrus may represent a morphological correlate to the enhanced ability of foodrestricted rats to acquire time /place associations is an attractive one, it must be regarded with caution. Indeed, both the involvement of the hippocampal formation in integration of spatio-temporal information [27,35] and the functional implications of the synaptic changes caused by restricted feeding in the dentate gyrus molecular layer [3] remain to be demonstrated. The present experiment was specifically designed to examine whether rats are able to use temporal cues in order to acquire time-of-day discrimination in a twochoice water escape spatial learning task and whether this ability is dependent on the activity of a circadian pacemaker entrained by timed restricted feeding. The results herein reported show that animals that were subjected to timed restricted feeding were capable of discriminating between two different escape platform positions corresponding to two different times of day, providing thus the first evidence that time-of-day discrimination can be acquired in the aversively motivated behavioral task. In contrast, animals that were fed ad libitum, even after a very extensive training, were unable to consult their internal clocks in order to discriminate between the two escape platform positions. Although we are still far from understanding the brain mechanisms subserving integration of spatial and temporal information, it appears that the feeding-entrainable circadian system is critically involved in cognitive processes associated with time/place learning.
Acknowledgements This work was supported by Fundac¸a˜o para a Cieˆncia e a Tecnologia (Unit 121/94; Projects SFRH/BPD/1583/ 2000 and PRAXIS/C/SAU/13186/1998).
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[32] Oliveira MH, Antunes-Rodrigues J, Leal AM, Elias LL, Moreira AC. Circadian variations of plasma atrial natriuretic peptide and corticosterone in rats with continuous or restricted access to food. Life Sci 1993;53:1795 /801. [33] Olton DS. Memory functions and the hippocampus. In: Seifert W, editor. Neurobiology of the hippocampus. New York: Academic Press, 1983:335 /74. [34] Pitsikas N, Algeri S. Deterioration of spatial and nonspatial reference and working memory in aged rats: protective effect of life-long calorie restriction. Neurobiol Aging 1992;13:369 /73. [35] Rawlins JN, Winocur G, Gray JA. The hippocampus, collateral behavior, and timing. Behav Neurosci 1983;97:857 /72. [36] Reebs SG. A test of time /place learning in cichlid fish. Behav Processes 1993;30:273 /82. [37] Roberts HJ, Wearden JH, Smart JL. Undernutrition of weanling and adult rats: effects on operant responding. Behav Brain Res 1983;10:287 /96. [38] Sandi C, Loscertales M, Guaza C. Experience-dependent facilitating effect of corticosterone on spatial memory formation in the water maze. Eur J Neurosci 1997;9:637 /42. [39] Schatz B, Beugnon G, Lachaud J-P. Time /place learning by an invertebrate, the ant Ectatomma ruidum . Roger Anim Behav 1994;48:236 /8. [40] Sharma VK, Chidambaram R, Subbaraj R, Chandrashekaran MK. Effects of restricted feeding cycle on the locomotor activity rhythm in the mouse Mus booduga . Physiol Behav 2000;70:81 /7. [41] Shors TJ. Acute stress rapidly and persistently enhances memory formation in the male rat. Neurobiol Learn Mem 2001;75:10 /29. [42] Stokkan KA, Yamazaki S, Tei H, Sakaki Y, Menaker M. Entrainment of the circadian clock in the liver by feeding. Science 2001;291:490 /3. [43] van Esseveldt KE, Lehman MN, Boer GJ. The suprachiasmatic nucleus and the circadian time-keeping system revisited. Brain Res Rev 2000;33:34 /77.
Research report
Restricted feeding facilitates time place learning in adult rats /
Nikolai V. Lukoyanov *, Pedro A. Pereira, Rui M. Mesquita, Jose´ P. Andrade Department of Anatomy, Porto Medical School, Alameda Prof. Hernaˆni Monteiro, 4200-319 Porto, Portugal Received 2 January 2002; received in revised form 6 February 2002; accepted 6 February 2002
Abstract Many species can acquire time-of-day discrimination when tested in food reinforced place learning tasks. It is believed that this type of learning is dependent upon the ability of animals to consult their internal circadian pacemakers entrained by various environmental zeitgebers, such as light /dark cycles and scheduled restricted feeding. In the present study, we examined, (1) whether rats can acquire time-of-day discrimination in an aversively motivated water maze task wherein an escape platform is located in one position in the morning and in another position in the afternoon; (2) whether time-of-day cues provided by the light- and feedingentrainable pacemakers may have divergent impacts upon the ability of rats to learn this task. Two groups of rats, both maintained on 12-h light:12-h dark cycle, were used; in one group, animals had free access to food, whereas in the other, they were subjected to a restricted feeding protocol (60% of food consumed by rats fed ad libitum, once daily). Despite the heightened difficulty of the task, food-restricted rats were apparently able to acquire associations between two different platform positions and two different times of day, as indicated by the fact that the percentage of discrimination errors in this group declined progressively, as a function of training, and stabilized at the level of 229/9%. In contrast, rats that were fed ad libitum, even after extensive training, failed to perform the task above level of chance. These data indicate that time /place learning is a universal, reward-nonspecific, cognitive phenomenon. They furthermore suggest that the ability of animals to integrate spatial and temporal information can be dependent on the access to timing stimuli provided by the feeding-entrainable circadian system. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Time /place learning; Timed hypocaloric diet; Two-choice water maze; Win-stay strategy; Win-shift strategy; Corticosterone; Circadian rhythms
1. Introduction Substantial evidence from behavioral studies indicates that animals are capable of organizing their daily activities, particularly those devoted to feeding, by using time of day as a discriminative temporal cue. For example, the ability to associate different food locations with different times of day has been described in ants [39], fish [36], birds [1], and in mice housed in seminaturalistic environment [14]. Laboratory studies have yielded similar results. It has been reported that rats can learn to press one lever for food in the morning and the other in the afternoon [4,7]. It was also found that rats can learn a time-of-day discrimination T-maze task in which they are required to search for food in one arm of
* Corresponding author. Tel.: 351-22-509-6808; fax: 351-22550-5640 E-mail address: [email protected] (N.V. Lukoyanov).
the maze during a morning training session and in the other during an afternoon training session [25,26,29]. However, it was also reported that rats learn more readily to initiate food-seeking behavior at the appropriate time than to associate availability of food at specific locations with times of day and that relatively small proportion of them (approximately 60%) are able to acquire a criterion of 90% correct choices in a true time/place learning task [25,26]. Although it seems obvious that time /place learning is based upon the ability of animals to integrate spatial information with temporal cues, both the nature and the origin of these temporal cues remain enigmatic. One possibility is that animals can consult their circadian pacemaker entrained by light /dark cycles and known to be located in the suprachiasmatic nucleus of the hypothalamus [21,43]. However, the observations that time-of-day discrimination is not disrupted by complete ablation of this nucleus and by housing animals in
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constant light argues against this hypothesis [28,29]. It has also been suggested that time-of-day cues are provided by a circadian pacemaker entrained by timed restricted feeding, the anatomical location of which is presently unknown [10 /12,28,29,40]. Thus, it can be hypothesized that maintenance of animals on ad libitum feeding regimen would result in a loss of their ability to learn time-of-day discrimination tasks. This possibility, however, has not yet been addressed. Indeed, all above cited laboratory studies on time /place learning were performed using appetitively motivated behavioral paradigms [4,7,8,25,26,29], a condition which does not permit the use of animals fed ad libitum. This problem can be circumvented by the employment of a nonappetitively motivated place learning task, such as the Morris water maze [30], which utilizes the ability of rodents to learn the position of a submerged escape platform in a large tank filled with opaque water. We introduced one additional variable to this task */time of day */by training rats to search for the platform in one position in the morning and in another position in the afternoon. In doing so, we sought to examine whether rats can consult their internal circadian pacemakers, entrained by either light /dark cycles alone [10,29] or in combination with timed restricted feeding [9,28], in order to associate different locations of the escape platform with different times of day.
2. Methods 2.1. Animals Sixteen male Wistar rats derived from the colony of the Gulbenkian Institute of Science (Oeiras, Portugal) were used in the present study. Animals were housed individually, and maintained under standard laboratory conditions (20 /22 8C, lights on from 07:00 to 19:00 h, free access to food and water). At 8 weeks of age, rats were randomly assigned to one of two groups with eight animals in each. Rats in the first group were fed ad libitum with a standard laboratory chow (Letica, Spain), supplemented with salts and vitamins (AL group), while rats in the other group were fed 60% of the amount of food consumed by AL animals (FR group [3]). These FR rats were fed once a day either at 08:00 or at 11:00 h, following a random schedule ensuring that the feeding procedure cannot be used by the animals as a discriminative cue. Both groups of rats were maintained on their respective feeding regimen until the end of the experiment. At 20 weeks of age, all rats were handled 5 min per day for 5 consecutive days and submitted to behavioral testing. During the period of handling, blood samples (300 ml) were collected from the dorsal vein of the tail at 08:00, 13:00, and 18:00 h. These samples were
used to analyze circadian fluctuations in the serum levels of corticosterone. The handling and care of the animals were conducted according to the European Communities Council guidelines in animal research (86/609/UE). 2.2. Serum corticosterone levels The blood samples were centrifuged, and the serum was separated and stored at /20 8C until the time of assay. Serum corticosterone levels were determined by radioimmunoassay using a kit from ICN Biomedicals. All samples were analyzed in a single run and the intraassay coefficient of variation was 5.2%. 2.3. Water maze apparatus The behavioral apparatus used in this study has been described in detail previously [3,23]. Briefly, it consisted of a black circular tank, 1.8 m in diameter and 0.5 m deep. The tank was located in a corner of a room containing extra-maze cues, i.e. three posters and desk with computer. The position of the pool and of the extra-maze cues was kept unchanged throughout the period of behavioral testing. The apparatus was filled with water at room temperature (219/1 8C) to a depth of approximately 35 cm. The water was made opaque by adding a nontoxic paint. The pool was divided, by imaginary lines, into four equal-size quadrants. A black circular escape platform, 10 cm in diameter, was located 2 cm below the surface of water. It was positioned in the center of one of the quadrants during all morning training sessions and in the center of the opposite quadrant during all afternoon sessions. The swim path was recorded by a computerized video-tracking system (EthoVision V1.90, Noldus, The Netherlands). 2.4. Procedure The procedure for the water maze testing was essentially as described previously [3,23] except that animals were required to learn the two different platform positions, one in the morning (beginning at 09:00 h) and the other in the afternoon (beginning at 18:00 h). For acquisition, rats were given one morning and one afternoon training sessions per day for 30 days. However, in order to reduce the possibility that the animals can perform the task using a simple session-to-session alternation strategy, one of each five consecutive sessions was randomly omitted. Therefore, a total of 25 morning and 25 afternoon training sessions was given. Each session consisted of one testing trial followed by three reinforcing trials. Each trial begun by placing the rat into the water facing the pool wall at one of four starting points that were used in a pseudorandom order so that each point was used once
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in each session. However, the first trials of each session were always given from one of two starting points equidistant to both platform positions. The starting points were the same for all animals. Each trial ended when the rat climbed onto platform. If the rats did not find the escape platform within 60 s, the experimenter guided them to the platform. After climbing onto platform, all rats were allowed to remain there for 15 s. The intertrial interval was 30 s. After the last trial of every session, the rats were towel dried and placed in a holding cage close to a heater for 15 min. The distances swum by rats to find the escape platform were analyzed. In addition, the numbers of discrimination errors, defined as swims through the area corresponding to the afternoon platform position during morning training and vice versa, were registered. Since on the second, third and fourth session trials animals could use the information gained on the anterior trials of that session, only the pathways derived from the first session trials (testing trials) were analyzed. One day after completion of the task, animals received a single 60-s probe trial in which the platform was removed from the pool. All rats were released from the same starting point equidistant to both former platform positions. The number of times the rats swum through the areas where the platform had been located in the morning and in the afternoon (platform crossings) and the percentage of time spent in the two training quadrants corresponding to the morning and afternoon platform locations were recorded. 2.5. Statistical analysis The serum corticosterone levels were analyzed using repeated measures ANOVA. The swim distances to find the platform in the morning and in the afternoon were averaged over blocks of five consecutive sessions each and analyzed using repeated measures ANOVA. The errors made in the discrimination between the two platform positions were averaged over blocks of ten sessions each (five consecutive morning plus five consecutive afternoon sessions) and analyzed using repeated measures ANOVA. The discrimination errors made by rats on the first sessions following the omission tests were averaged over consecutive blocks of one morning session and one evening session. The time-of-day discrimination errors made by rats during the last training sessions, separately in the morning and in the afternoon, were averaged over blocks of five last morning and five last afternoon sessions each and analyzed using two-way ANOVA. The measures derived from the probe trial were analyzed using one-way ANOVA. Newman /Keuls post hoc test was used where appropriate. The data are expressed as the mean9/ S.E.M. Differences were considered as significant at the P B/0.05 level.
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3. Results 3.1. Serum corticosterone levels Serum levels of corticosterone found in blood samples collected at 08:00, 13:00 and 18:00 h are shown in Table 1. In rats that were fed ad libitum, the corticosterone levels were lowest in the morning and reached their highest values late in the afternoon. In food-restricted rats the highest concentrations of the hormone were found in blood samples collected in the morning and in the afternoon, while they were lowest in samples collected at 13:00 h. Although there was no significant main effect of feeding regimen on the corticosterone levels, ANOVA revealed a significant effect of sampling time (F2,28 /5.53, P B/0.01) as well as a significant interaction between feeding regimen and sampling time (F2,28 /9.40, P B/0.001). Post hoc tests indicated that corticosterone levels in AL rats were significantly higher at 18:00 than at 08:00 and at 13:00 h (P B/0.01). Likewise, in FR animals, corticosterone levels were significantly higher in samples collected at 08:00 and at 18:00 h than in those collected at 13:00 h (P B/0.001 and P B/0.01, respectively). Finally, the levels of corticosterone measured at 08:00 h were significantly higher in FR rats than in AL rats (P B/0.001). No significant differences between FR and AL groups were found when comparing serum corticosterone concentrations in samples collected at 13:00 and 18:00 h. 3.2. Swimming distances Distances swum by rats to find the hidden platform in the morning and in the afternoon are presented in Fig. 1. The overall repeated-measures ANOVA showed that food-restricted rats and rats fed ad libitum progressively improved their ability to locate both platform positions over the entire course of training (F4,112 /91.12; P B/ 0.0001). Although there were no significant effects of feeding regimen and time of day on this measure, ANOVA detected a significant interaction between these two variables (F1,28 /9.54; P B/0.005). Pairwise comparisons revealed that, in the afternoon, rats from AL group swam shorter distances than did FR animals Table 1 Serum levels of corticosterone (ng/ml) in blood samples collected from rats fed ad libitum (AL) and food-restricted (FR) rats at 08:00, 13:00 and 18:00 h Groups
08:00 h
13:00 h
18:00 h
AL FR
108923 242933**
113916 110918
195915* 181918*
Values are presented as mean9S.E.M. *, P B 0.01 vs. 08:00 h in AL group and vs. 13:00 h in both groups. **, P B 0.001 vs. 08:00 h in AL group and vs. 13:00 h in both groups.
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Fig. 1. The distances (in centimeters) swum by rats fed ad libitum (AL) and by food-restricted (FR) rats to reach a hidden platform in the morning and in the afternoon for each block of five consecutive training sessions. Note that, at the beginning of the training, rats in AL group located the platform faster in the afternoon than in the morning. *, P B 0.05 vs. AL group in the morning and FR group in the afternoon.
(F1,14 /22.85; P B/0.001). Moreover, AL rats performed significantly better in the afternoon than in the morning (F1,14 /9.91; P B/0.01). Post hoc tests showed, however, that this effect was significant only at the beginning of acquisition (P B/0.05 for the first and second session blocks) and, after 10 days of training, had became undetectable (Fig. 1).
3.3. Discrimination errors The percentages of errors made by rats in first session trials, averaged over blocks of ten (five morning plus five afternoon) consecutive sessions each, are shown in Fig. 2. The overall repeated-measures ANOVA applied on these data revealed a significant effect of session blocks (F4,56 /5.26; P B/0.01) indicating that acquisition of the task was associated with a significant decrease in the percentage of errors made in initial trials. This decrease, however, was not uniform in FR and AL groups as indicated by a significant main effect of feeding regimen on the error level (F1,14 /9.59; P B/ 0.01). In fact, only animals in FR group significantly improved their performance over the course of training (F4,35 /3.65; P B/0.01), whereas the corresponding decrease in the percentage of errors made by AL rats did not reach the level of significance (F4,35 /1.92; P / 0.13). At the end of training, FR rats performed significantly above chance level (P B/0.05), making at most 25% of incorrect choices. In contrast, animals in AL group, even after extensive training, failed to perform above chance level, making significantly greater number of errors relative to FR rats (P B/0.05 for the session blocks 3 /5).
Fig. 2. The percentages of incorrect choices for each block of ten training sessions (five consecutive sessions given in the morning plus five consecutive sessions given in the afternoon). The percentages of errors made by rats on the first sessions following the omission tests, averaged over consecutive blocks of two sessions (one given in the morning and the other in the afternoon), are indicated by solid symbols. Note a progressive decrease in the number of errors made by food-restricted (FR) rats. In contrast, animals fed ad libitum (AL) failed to succeed in this task, performing at close to chance level throughout the entire course of training. *, P B 0.05 vs. AL group.
The percentages of discriminative errors made by rats on the first sessions following the omitted sessions, averaged over blocks of one morning and one evening session each, are shown in Fig. 2. Repeated-measures ANOVA failed to reveal any significant difference between the percentages of errors made by rats on these sessions and on the sessions that were not preceded by the omissions, indicating that the performance of rats in either group was not disrupted by the omission tests. The percentages of errors in initial trials made by rats in each group during the last five training sessions, separately in the morning and in the afternoon, are presented in Fig. 3. ANOVA applied on these data revealed a significant effect of feeding regimen (F1,14 /
Fig. 3. The percentages of incorrect choices made by rats in the morning and in the afternoon averaged over the last five training sessions. Note that FR animals had similar error scores in the morning and in the afternoon, whereas rats fed AL performed particularly poorly in the morning, making far fewer errors in the afternoon. *P B 0.01 vs. AL group in the afternoon and FR group both in the morning and in the afternoon.
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7.06; P B/0.01), a significant effect of time of day (F1,14 /9.63; P B/0.05) and a significant interaction between the two variables (F1,14 /13.69; P B/0.01). Inspection of these data indicated that AL rats performed particularly poorly in the morning (809/8% of incorrect responses), making far fewer errors in the afternoon (149/5%; P B/0.01 for the difference). Unlike AL rats, animals in FR group had similar error scores during the morning and afternoon training sessions (199/13 and 259/9%, respectively). In addition, post hoc
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tests indicated that, whereas FR rats significantly outperformed AL rats during the morning training sessions (P B/0.01), the percentages of errors made in the afternoon did not significantly differ between the groups. 3.4. Probe trial The results of the probe trial are shown in Fig. 4. In this test, rats from FR and AL groups spent an identical proportion of time swimming in either of the two training quadrants corresponding to the morning and afternoon positions of the platform. Likewise, the numbers of times the animals crossed the areas where the escape platform had been located during the morning and afternoon sessions (platform crossings) were highly similar in either group. ANOVA failed to detect any significant effect of feeding on these parameters.
4. Discussion
Fig. 4. Performance of rats fed AL and FR rats on the probe trial. (A) Path taken by a representative rat in AL group. Paths like this were taken by rats in both groups. The two positions where the platform had been located during training sessions given in the morning and in the afternoon are indicated. Also shown are (B) number of times the rats crossed the former platform positions (platform crossings) and (C) percentage of time spent swimming within the two training quadrants. No significant differences between groups were found on this test. Furthermore, in either group, no differences between the measures corresponding to the two different platform positions were detected.
One of the main findings of the present experiment is that rats that were subjected to timed food restriction were apparently able to learn the time-of-day discrimination version of the water escape task, as indicated by the progressive decrease in the number of incorrect choices made by these animals over the course of training. This observation fits nicely the results of several previous studies showing that food-restricted rats can learn to use temporal cues to press a correct lever for food [4] or to find food in a T-maze [25,26,29]. Importantly, current data extend the previously reported findings revealing the capacity of food-restricted rats to acquire time-of-day discrimination not only in the appetitively motivated tasks but also when they are required to learn spatial locations of escape routes. Considered together, these results thus suggest that time/place learning is a universal, reward-nonspecific, cognitive phenomenon. On the other hand, we have additionally found that time-of-day discrimination in the water maze is quite difficult for rats to learn. Indeed, the animals approached the asymptotic performance only after they were given as many as 200 trials (taking into account the reinforcing trials); even so, the level of correct choices did not exceed 80%. For comparison, learning of the classical Morris water maze, in which the escape platform is located in a constant position, usually requires 20 /30 training trials [3,23,30]. It has been previously reported that rats have difficulty acquiring time-of-day discrimination in appetitively motivated tasks [8,25,26]. Another principal finding of this study is that, unlike food-restricted rats, animals that were fed ad libitum failed to acquire the temporal discrimination component of the task. This finding is illustrated by the fact that,
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even after a very extensive training, the number of correct choices made by these rats in first session trials remained at chance level. Yet, the distances swum to find the platform at either position did not differ between groups, except during the initial phase of training. Furthermore, the measures derived from the probe trial, that is platform crossings and percentages of time spent in training quadrants, were identical in AL and FR groups, suggesting that animals in both groups had comparable spatial learning and memory abilities. These data indicate that the low percentage of correct choices shown by AL rats is not related to spatial learning deficits, but rather, reflects their inability to associate spatial information with temporal cues. The observation that rats in FR group were able to acquire time-of-day discrimination, while those in AL group were not, can imply that animals in these two groups had access to different sets of temporal cues, provided probably by different circadian oscillators. In support of this view, we found that, whereas in animals that were fed ad libitum the serum levels of corticosterone were elevated only late in the afternoon, in those that were maintained on restricted feeding regimen the corticosterone levels were in addition prominently increased in the morning. The fact that the two peaks of corticosterone release were coincident either with the onset of nocturnal activity (in both groups) or with the anticipation of feeding (in FR group) indicates that they were entrained, respectively, by light /dark cycles [13] and by the scheduled daily meal [18,32,42]. Thus, taking into account that only rats from FR group performed the task above chance level, it is possible to assume that the ability to acquire time-of-day discrimination is largely dependent upon the access to temporal information provided by the food-entrainable circadian oscillator [25,26,28,29]. When analyzing the numbers of incorrect choices made by rats during the last 5 days of training, we unexpectedly found that the animals in AL group were far more precise in the afternoon (14% of errors) than in the morning (80% of errors). In other words, these animals adopted a perseverative behavioral response, known also as a win-stay strategy [15], */always to inspect first the presence of the platform in its afternoon position*/and applied it for both daily sessions. In contrast, animals in FR group adopted a win-shift behavioral strategy required to correctly perform the two-choice water escape task, as indicated by the observation that these rats were able to use two different navigation responses corresponding to the two different platform positions. The finding that AL rats favored to use the afternoon platform position as an escape route of first choice can imply that, during training, they gained more spatial knowledge in the afternoon, probably, due to the high circulating levels of corticosterone known to facilitate spatial learning [22,31,38,41], than in
the morning, when the levels of corticosterone were relatively low. This is consistent with the observation that, at the beginning of training, these animals learned the afternoon position of the platform faster than its morning position. However, such an interpretation conflicts with our finding that, at the end of training, the amount of spatial information acquired by AL rats was the same for both platform positions, as indicated by the distances swum during the last training sessions as well as by the results of the probe trial. Thus, while perhaps accounting for the different rates of initial learning shown by AL rats in the morning and in the afternoon, this hypothesis does not explain why the animals consistently used the win-stay strategy throughout the entire period of training. Given these considerations, it seems more likely that the simple win-stay strategy employed by rats in AL group is associated with their inability to use time of day as a discriminative stimulus and, therefore, might have for them an adaptive value. It can also be suggested that the difference between the two groups in the performance on the time /place task is related to the beneficial effect of food restriction on the cognitive abilities of rats. This hypothesis is strongly supported by the evidence indicating that restricted feeding enhances learning and memory in aged cognitively impaired rodents [19,20,34] and improves behavioral outcome following different types of brain lesions [6,16]. Surprisingly, the beneficial effects of restricted feeding on cognitive processes in healthy adult rats have never been convincingly demonstrated, despite the fact that different types of learning and memory were tested using different behavioral paradigms, such as the radial maze [17], Morris water maze [3,24], and operant conditioning [37] and passive avoidance [3] tasks. Furthermore, it has been recently shown, in different laboratories [3,24], that restricted feeding does not alter the performance of adult rats on the variable platform position version of the Morris water maze, a task known to rely on the ability of rats to use both the win-stay and the win-shift strategies. Nonetheless, it remains possible that the enhanced time /place learning shown by food-restricted rats in our experiment is associated with beneficial effects of restricted feeding on some other types of behavioral processes, which were not properly assessed in the above-cited studies. Future comparative experiments on the effects of this feeding regimen on animal behavior will help to clarify this issue. Taking into account the current data as well as the findings of previous studies [4,5,25,26,29], it appears that access to temporal information provided by the food-entrainable circadian system is indeed essential for the ability of animals to acquire time /place associations. However, it remains unknown how this temporal information is coupled with the spatial one and which
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regions of the brain are involved in this process. There is now strong evidence that the hippocampal formation plays a critical role both in the processing of spatial information and in associative learning [33]. Therefore, it is theoretically possible that the integrative cognitive processes underlying time/place learning are also subserved by hippocampal neuronal circuits. Interestingly, we have recently found that maintenance of rats on a hypocaloric diet similar to that used in the present experiment produces a 15% increase in the total number of synapses in the molecular layer of the dentate gyrus [3]. Bearing in mind that the vast majority of hippocampal afferents terminate in the dentate gyrus [2], it can be speculated that the synaptic changes in this area are somehow related to the increased flow of temporal information arriving to the hippocampal formation from the food-entrainable circadian system. Admittedly, although the idea that the increase in the number of synapses in the dentate gyrus may represent a morphological correlate to the enhanced ability of foodrestricted rats to acquire time /place associations is an attractive one, it must be regarded with caution. Indeed, both the involvement of the hippocampal formation in integration of spatio-temporal information [27,35] and the functional implications of the synaptic changes caused by restricted feeding in the dentate gyrus molecular layer [3] remain to be demonstrated. The present experiment was specifically designed to examine whether rats are able to use temporal cues in order to acquire time-of-day discrimination in a twochoice water escape spatial learning task and whether this ability is dependent on the activity of a circadian pacemaker entrained by timed restricted feeding. The results herein reported show that animals that were subjected to timed restricted feeding were capable of discriminating between two different escape platform positions corresponding to two different times of day, providing thus the first evidence that time-of-day discrimination can be acquired in the aversively motivated behavioral task. In contrast, animals that were fed ad libitum, even after a very extensive training, were unable to consult their internal clocks in order to discriminate between the two escape platform positions. Although we are still far from understanding the brain mechanisms subserving integration of spatial and temporal information, it appears that the feeding-entrainable circadian system is critically involved in cognitive processes associated with time/place learning.
Acknowledgements This work was supported by Fundac¸a˜o para a Cieˆncia e a Tecnologia (Unit 121/94; Projects SFRH/BPD/1583/ 2000 and PRAXIS/C/SAU/13186/1998).
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