Behavioural Processes 44 (1999) 287 – 299

Rats are reluctant to use circadian timing in a daily time–place task Jason A.R. Carr *, Donald M. Wilkie Department of Psychology, Kenny Building, 2136 West Mall, Uni6ersity of British Columbia, Vancou6er, BC, Canada V6T 1Z4 Received 15 April 1998; received in revised form 28 July 1998; accepted 31 July 1998

Abstract On daily time–place learning tasks animals can work for food at different spatial locations during sessions at different times of the day. In previous experiments rats tracked this pattern of food availability with ordinal timing—they learned to respond at the locations in the correct order each day. In contrast, pigeons used circadian timing. In this experiment rats received a mixture of morning session only days, afternoon session only days, and morning and afternoon session days. Under these conditions ordinal timing had low predictive ability, but circadian timing was potentially perfectly predictive of the location of food availability. We thought this procedural change might encourage rats to use circadian timing. However, we found little evidence that rats can use time of day information to track this daily spatiotemporal pattern of food availability. These results are suggestive of differences in the use of circadian clock consultation by rats and pigeons. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Circadian rhythm; Foraging behaviour; Rats; Species difference; Time – place behaviour; Timing

1. Introduction A variety of organisms exploit food sources that display circadian spatiotemporal patterns of availability (for a review see Carr and Wilkie, 1997a). For example, flight-hunting in kestrels tends to coincide in time with the peaks in the surface activity of their main prey, the common * Corresponding author. Tel.: +1-604-8224650; fax: +1604-8226923; e-mail: [email protected]

vole (Rijnsdorp et al., 1981). Similarly, Wilkie et al. (1997) found that the number of scavenging birds present at two outdoor locations where people eat lunch reached a maximum just before the midday peak in the number of people present. Field reports of this nature have inspired several laboratory investigations of daily time–place learning (Wilkie, 1995). In many of these studies animals reliably anticipated changes in the location of food availability over the course of the day. This finding suggests that the animals used

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an endogenous timing system to track, and thereby exploit, the daily spatiotemporal pattern of food availability. Recently attention has been focused on characterizing the timing systems that animals use to solve this potentially fundamental problem. In daily time–place tasks the spatiotemporal pattern of food availability has a 24-h periodicity. Consequently, it is reasonable to suspect that animals might use circadian timing to track the location of food availability in these tasks. Attempts to test this notion have capitalized on a key attribute of circadian timing. Time of day information is thought to be provided by the phase angle of an endogenous circadian clock. Therefore, time–place behaviour that is controlled by circadian timing should display the operating characteristics of biological circadian systems. One key operating characteristic of circadian systems is entrainment. Certain daily exogenous cues (Zeitgebers) entrain, or adjust, circadian oscillators so that each oscillator holds a fixed phase relationship with it’s entraining cue. Two major Zeitgebers are the light – dark cycle (LD) and brief daily periods of food availability. In rats and pigeons these Zeitgebers appear to entrain separate circadian pacemakers (Rosenwasser and Adler, 1986; Abe and Sugimoto, 1987; Phillips et al., 1993; Mistlberger and Rusak, 1994). When a Zeitgeber is removed (e.g. an animal is held in constant dim light (LL)), endogenous oscillators persist but often attain a periodicity slightly different from 24 h, a phenomena called ‘free-run’. This self-sustaining rhythmicity reflects the inherent periodicity of the circadian pacemaker driving the oscillatory system. If a Zeitgeber is phase advanced (occurs earlier), or phase delayed (occurs later), the oscillator it controls drifts in the direction of the phase shift and re-entrains to the Zeitgeber. Saksida and Wilkie (1994) tested pigeons in a daily time–place task. The birds were transported to a large, transparent testing chamber twice per day; once between 09:00 and 10:00 h and a second time between 15:30 and 16:30 h. For each bird, pecks to one key produced grain in morning sessions, and pecks to a different key produced

grain during afternoon sessions. Responses recorded during a brief nonrewarded period at the beginning of each session served as a measure of the pigeons’ ability to anticipate the location of food availability. With experience, the pigeons learned to peck primarily the correct key during morning and afternoon sessions. When either morning or afternoon sessions were occasionally skipped, the pigeons still were able to anticipate the location of food availability at the beginning of the next session. This finding suggests that the birds were not using a sessionto-session alternation strategy to track the location of food availability. Lighting manipulations (e.g. constant dim LL and LD phase shifts) initially had no effect. However, after several days the birds’ time–place behaviour became disrupted as the phase relationship between the birds’ circadian clocks and the spatiotemporal pattern of food availability was weakened. Taken together, these results suggest that pigeons used time of day information provided by a light-entrained circadian clock to track this daily pattern of food availability. There is evidence that animals other than pigeons also use circadian timing to track daily spatiotemporal patterns of food availability. For example, there is evidence for the use of circadian timing in daily time–place tasks by other species of birds including garden warblers (Biebach et al., 1989, 1991), weavers (Falk et al., 1992), and starlings (Wenger et al., 1991). There is also some evidence for the use of circadian timing in daily time–place tasks by fish (Reebs, 1996), and ants (Schatz et al., 1994). We recently investigated daily time–place learning in rats and obtained a very different result (Carr and Wilkie, 1997b). With the exception of a change in the response manipulandum used (levers were substituted for the pecking keys), our testing procedures were the same as those used with pigeons by Saksida and Wilkie (1994). Rats clearly learned to respond on the correct lever during morning and afternoon test sessions. In the morning sessions that followed occasional skipped afternoon sessions, the rats continued to expect food at their morning levers. However in the afternoon sessions that followed occasional

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skipped morning sessions, the rats incorrectly expected food at their morning levers. This outcome suggests two conclusions: (1) receiving food at their morning levers, and not the passage of time, was necessary for the rats to anticipate the location of food availability in afternoon sessions; and (2) receiving food at their afternoon levers was not necessary for the rats to anticipate the location of food availability during the following morning session. Evidently the rats did not use the phase angle of a circadian clock, the accumulated value of an interval timer started by a daily event such as colony light onset or awakening, or a session-to-session alternation strategy, to discriminate between morning and afternoon sessions. Instead, our rats’ time – place behaviour appeared to be based primarily on a representation of the order in which food was available at their two feeding sites each day. The rats pressed one lever during their first session of each day, and pressed a second lever during their second session of each day. We have recently obtained very similar results with rats tested on a three session per day daily time –place task (Carr et al., in review). With experience rats were able to anticipate the location of food availability during morning, midday, and afternoon sessions. During the morning sessions that followed occassional skipped afternoon sessions, the rats correctly preferred their morning levers. However, during the midday sessions that followed occassional skipped morning sessions, the rats incorrectly preferred their morning levers. Similarly, during the afternoon sessions that immediately followed occasional skipped midday sessions, the rats incorrectly preferred their midday levers. Evidently the rats were pressing one lever during their first session of each day, pressing a second lever during their second session of each day, and pressing a third lever during their third session of each day. We have argued that this knowledge of the temporal order of a set of events within a period of time constitutes ordinal timing (Carr and Wilkie, 1997a,b). In our original daily time – place task both ordinal and time of day information could, in theory, perfectly predict the location of food: Each day food availability followed a reliable spatiotempo-

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ral sequence, and each day morning and afternoon sessions occured at the same time of day. Given this, our previous results obtained with pigeons and rats may reflect a quantitative difference in the way that these animals weight ordinal and time of day information in our daily time– place task. Perhaps both animals process circadian and ordinal information, but rats preferentially use ordinal information and pigeons preferentially use circadian information. On the other hand, rats may be unable to track daily spatiotemporal patterns of food availability with a circadian clock. Two groups of researchers have concluded that rats can use circadian information to track daily spatiotemporal patterns of food availability. Boulos and Logothetis (1990) held rats individually in circular light-tight chambers. Food was provided at one lever at one time of day and at a second lever at a second time of day. Most rats displayed increases in lever pressing in anticipation of both daily meals, and slightly less than half of their rats anticipated the location of both their morning and afternoon meals. However, this demonstration does not prove that rats used circadian timing to track the location of food availability as no tests were conducted to rule out the rats’ use of a session-to-session alternation strategy. Mistlberger et al. (1996) reported stronger evidence that rats can use time of day information to track food availability in a daily time–place task. Their rats were held in a 12 h LD cycle. Each day the rats received two test sessions on a T-maze which had a response lever and hopper mounted on two arms. Intact rats and rats with lesions of the suprachiasmatic nuclei (SCN) were tested. The SCN is the site of the light-entrained circadian clock in mammals (for a review see Klein et al., 1991). For all the rats, responses on one lever produced food in morning sessions, and responses on a second lever produced food in afternoon sessions. Both the intact and SCN-lesioned rats learned to press the correct lever during morning and afternoon sessions. Mistlberger et al. (1996) then skipped morning sessions, afternoon sessions, and a block of three consecutive test sessions. In the

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sessions following the majority of the skipped sessions their rats clearly anticipated which lever would provide them with food. This rules out the rats’ use of ordinal timing, or a session-to-session alternation strategy. Mistlberger et al. then inverted the rats’ LD cycle twice and held the rats in constant dark (DD) once. For the most part the rats’ time–place behaviour was unaffected by these lighting manipulations. This outcome makes the rats’ use of a light-entrained circadian clock or interval timing from colony light onset or awakening unlikely. Given these results, Mistlberger et al. concluded that their rats likely discriminated between morning and afternoon sessions by consulting the phase angle of a food-entrained circadian clock. Given the findings of Mistlberger and coworkers, we thought that perhaps our original daily time –place task somehow failed to draw on rats’ ability to use circadian timing to track a daily spatiotemporal pattern of food availability. Consequently, we modified our task with the hope of making it a more sensitive measure of rats’ circadian timing ability. In the modified task rats received morning session only days, afternoon session only days, and morning and afternoon session days. These three types of training days were given in a random order. This modified daily time –place task did not present rats with a reliable spatiotemporal sequence of food availability each day. Therefore, the location of food availability could not be predicted reliably with ordinal information. On the other hand, circadian phase information still perfectly predicted the location of food availability. We reasoned that this modified spatiotemporal pattern of food availability might encourage rats to display a previously untapped ability to use circadian timing to track the location of food availability in our daily time –place task.

2. Experiment 1: acquisition training

2.1. Materials and methods 2.1.1. Animals We tested four male hooded Long Evans rats

obtained from Charles River (St. Constant, Que., Canada). At the beginning of the experiment the rats were experimentally naive, approximately 90 days old, and weighed between 400 and 420 g. The animals tested were of the same age (at the beginning of testing), strain, and sex as those tested by Carr and Wilkie (1997b). The rats were maintained at a minimum of 90% of their freefeeding weight (adjusted for age) and received free access to water except during the experimental sessions. The rats were fed 45-mg Noyes A/I Reward Pellets (P.J. Noyes Company, Lancaster, NH) during experimental sessions and standard Rat Lab Diet (PMI Feeds, St. Louis, MO) during post-session feedings. The rats lived individually in opaque plastic cages lined with Bed-o’cobbs bedding material (The Andersons, Maumee, OH). They were periodically given paper products to build nests. Colony light onset was at 07:30 h and light offset was at 19:30 h, producing a 12 h LD cycle. Daily behavioural enrichment is a standard component of our animal care program. Consequently, the rats received daily enrichment sessions during which they could interact socially, explore, and manipulate objects. Throughout these experiments the rats were cared for in strict accordance with Canadian Council on Animal Care guidelines.

2.1.2. Apparatus The rats were tested in a large transparent Plexiglas chamber (40×40×42 cm). The chamber was located on a bench in a small (3× 2 m), well-lit testing room. The chamber and testing room were the same as those used by Carr and Wilkie (1997b). The floor of the chamber was covered with Bed-o’cobbs bedding material. From within the chamber the rats could view a variety of distal spatial cues including a PC and monitor, a door frame and door, and various geometric shapes cut from construction paper that were stuck to the walls of the room. A lever was centered horizontally on each of the four walls of the testing chamber, 10 cm above the chamber floor. A brass food cup was

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mounted adjacent to each lever. Lever presses were recorded by the closure of a microswitch mounted on each lever. Four pellet hoppers (Model No. D700, Scientific Prototype Manufacturing Corporation, New York) were mounted on the top of each test chamber. When operated, the hoppers dispensed 45-mg Noyes A/I Reward Pellets into the food cup mounted adjacent to their associated lever. A small covered cue light (24 V) was mounted above each lever. Data collection and equipment control were carried out by a C + + program running on a nearby networked PC.

2.1.3. Procedure Aside from the specified changes in the scheduling of sessions, the training procedures were identical to those used by Carr and Wilkie (1997b). At the beginning of training the rats were exposed individually to the testing chamber. During these 30-min sessions every lever press delivered a reward pellet into the adjacent food cup. After six of these sessions all the rats were steadily pressing all four levers for food. Reward pellets were then provided on a variable ratio (VR) schedule. The VR was gradually increased until all the rats were pressing steadily on a VR16. Acquisition training then began. Prior to each session the rats were transported as a group to the enrichment chamber. They were then taken one at a time, and in a random order, to the testing chamber. In the testing chamber each rat could press for reward pellets on one lever during morning sessions and on a different lever during afternoon sessions. Lever assignment was counterbalanced across the rats. A session began when a rat pressed any lever. This turned on the four cue lights. The onset of the cue lights was followed by a 10-s ‘time-out’ period. During the time-out period responses were not recorded nor did they have any programmed consequence. The time-out period was followed by a period of variable length (range 4– 40 s) during which responses on all four levers were recorded but no reward pellets were given. Anticipatory responses recorded during this nonrewarded period were used to infer where the rats expected food to be available. This expectation was expressed in the

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form of a mean percent of all anticipatory responses score for each of the three types of levers: (1) the correct lever in that session (Corr); (2) the correct lever during sessions at the alternate time of day (Alt); and (3) the two incorrect levers that never provided food (Inc). This score was calculated for each type of lever by dividing the number of anticipatory responses on the lever(s) by the total number of anticipatory responses on all the levers, and then multiplying this ratio by 100. These three scores were calculated for each rat for each session. The initial nonrewarded period was followed by a 10-min rewarded period during which the designated lever provided reward pellets on a VR16. At the end of each session the cue lights were turned off and the rat was transported back to the enrichment box. After all four rats were tested they were returned to their home cages where they received a post-session feeding. The rats received their post-session feedings an average of 20 min (range 2–38 min) after the end of their experimental session. One or 2 days per week were rest days. During rest days the rats remained in their home cages and were fed at one of their usual post-session feeding times (i.e. at 10:30 or 16:30 h). The time of feeding on rest days was determined according to a random schedule. During Experiment 1 rats received morning session only days, afternoon session only days, and morning and afternoon session days. These three types of training days were given in a random order. As a result of this session scheduling procedure the rats received morning sessions that were preceded in time by another morning session (designated morning/morning sessions), and morning sessions that were preceded in time by an afternoon session (morning/afternoon sessions). Similarly, the rats received afternoon sessions that were preceded in time by another afternoon session (afternoon/afternoon sessions), and afternoon sessions that were preceded in time by a morning session (afternoon/morning sessions). During Experiment 1, one-third of all morning sessions were morning/morning sessions, and the remaining two-thirds were morning/afternoon sessions. Similarly, one-third of all afternoon sessions were afternoon/afternoon sessions, with the

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remaining two-thirds being afternoon/morning sessions. As sessions were scheduled according to a random schedule, these proportions varied from week to week over training.

2.2. Results Fig. 1 presents the rats’ overall mean Corr scores (for blocks of seven sessions) during the morning and afternoon sessions of acquisition training. During Block 1 the rats’ overall mean Corr scores for morning and afternoon sessions were both roughly 25%. This indicates that initially the rats did not know which levers would provide them access to food. If the rats were able to learn which two levers provided them food, but were unable to learn when to respond on each of the levers, they would be expected obtain a mean Corr score of 50%. Consequently, a Corr score of 50% was adopted as a conservative estimate of chance performance. Over the following weeks the rats’ overall mean Corr scores increased, first passing 50% in morning and afternoon sessions during Block 6. The rats’ Corr scores slowly increased with additional training, never falling below 50%, but failing to consistently exceed 75%. Acquisition training was extended over 21 blocks of morning and afternoon sessions (305 testing

Fig. 1. Overall mean Corr scores (for blocks of seven sessions) during the 21 weeks of aquisition training for Experiment 1. The dashed line indicates an initial chance level of 25%, and the solid line indicates a conservative final chance level of 50%. Corr=the correct lever in that session.

days). At this point it was evident that the rats’ discrimination performance had reached an asymptote. Inspection of the acquisition curves suggests that the rats’ discrimination performance did not differ in morning and afternoon sessions. These observations were confirmed by the results of a repeated-measures ANOVA performed on the mean Corr scores. Session Time (morning or afternoon) and Training (Block 1 through Block 21) served as repeated measures. This analysis revealed a significant effect of Training, [F(20,60)=10.95, PB0.001)], a non-significant effect of Session Time, [F(1,3)= 0.74, P B 0.46], and a non-significant Training×Session Time interaction, [F(20,60)B 1, P B 0.99]. Clearly the rats’ discrimination performance increased over the course of training, but was their final steady-state discrimination performance consistently better than that expected due to chance alone? The rats’ overall mean Corr scores during morning and afternoon sessions were highest during Block 16 of training [84.39 (S.E.M.= 4.66) and 76.11 (S.E.M.= 2.92), respectively]. After Block 16 the rats’ morning and afternoon overall mean Corr scores remained stable. Blocks 16 through 21 were therefore designated the acquisition asymptote period. Each rat’s mean morning and afternoon Corr score during the acquisition asymptote period was computed. The overall mean Corr score for the acquisition asymptote period was found to be 76.41 (S.E.M.= 4.04) for morning sessions, and 71.83 (S.E.M.= 4.02) for afternoon sessions. Both of these overall mean Corr scores are significantly greater than a conservative estimate of chance performance, [one sample t-test for morning sessions, t(3)= 6.54, PB 0.008, and for afternoon sessions t(3)= 5.43, PB 0.013]. When an overall measure of discrimination performance is used, the rats appear to have performed at above chance levels in the modified daily time–place task. However, closer inspection of these data suggested that this summary analysis is not representative of the rats’ performance in all types of sessions. During the 6-block long acquisition asymptote period the rats’ overall mean Corr scores only exceeded chance during three blocks (Blocks 16, 17, and 19) of morning sessions and two blocks (Blocks 16 and 18) of

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Fig. 2. Overall mean percent ( +S.E.M.) of all anticipatory responses that were on the rats’ Corr, Alt, and Inc levers during the acquisition baseline period of Experiment 1, for morning/morning, morning/afternoon, afternoon/afternoon, and afternoon/morning sessions. The solid line indicates a conservative final chance level of 50%. Corr =the correct lever in that session; Alt = the correct lever in the alternate daily session; Inc =the two levers that never provided food.

afternoon sessions [one sample t-tests, all t(3)’s \ 6.16, all P’s B 0.05]. Further investigation revealed that these three blocks of morning sessions and two blocks of afternoon sessions had the highest proportions of morning/afternoon and afternoon/morning sessions, respectively during the acquisition asymptote period. This observation was examined quantitatively. We calculated each rat’s mean Corr, Alt, and Inc score during morning/morning, morning/afternoon, afternoon/afternoon, and afternoon/morning sessions during the acquisition asymptote period. An overall mean Corr, Alt, and Inc score was then computed for each of the four types of sessions. These overall means (+ S.E.M.) are presented in Fig. 2. The rats clearly preferred their correct lever during all types of morning and afternoon sessions. However, their performance only reliably exceeded chance during the sessions that were preceded in time by a session at the alternate time of day (i.e. morning/afternoon and afternoon/morning sessions), [one sample t-test for morning/afternoon sessions, t(3) =5.01, P B 0.016, and for afternoon/morning sessions t(3) = 8.57, PB0.004, all other t(3)’s B1.98, P’s \0.14].

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The rats’ decreased discrimination performance in morning/morning and afternoon/afternoon sessions was almost entirely due to an increased preference for their Alt levers during these sessions. The decrease in the overall mean Corr score for morning/morning and afternoon/afternoon sessions was mirrored by an increase of almost exactly the same magnitude in the corresponding overall mean Alt score (for morning/morning sessions decrease in overall mean Corr score= 10.19 and increase in overall mean Alt score= 10.34; for afternoon/afternoon sessions decrease in overall mean Corr score=13.97, and increase in overall mean Alt score =13.59). Therefore, the rats’ decreased discrimination performance in morning/ morning and afternoon/afternoon sessions can be attributed to an increased preference for the lever that provided them food during the afternoon and morning, respectively.

2.3. Discussion With extensive training rats were able to perform at above chance levels in this modified daily time–place task when a relatively course measure of performance (mean Corr scores based on the average of seven sessions) was examined. However, finer-grained analyses found that the rats were only able to anticipate reliably the location of food availability during sessions that were preceded in time by a session at the alternate time of day. For example, for the rats to anticipate reliably the location of food availability during morning sessions, their preceding test session had to have been during the afternoon. It is difficult to imagine why an animal using the phase angle of a circadian clock to discriminate between morning and afternoon sessions would be affected in this manner by the time of day of previous test sessions. Consequently, these data provide no clear evidence that rats are able to use endogenous time of day information to track a daily spatiotemporal pattern of food availability. Instead, the rats’ decreased discrimination performance in morning/morning and afternoon/afternoon sessions suggests that their meager ability to predict the location of food availability was based, in part, on a session-to-ses-

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sion alternation strategy. When the rats received food at their morning levers they expected food next at their afternoon levers, and when they received food at their afternoon levers they expected food next at their morning levers1. It is important to stress that this session-to-session alternation strategy is not the same as the ordinal timing strategy that rats used in the original daily time–place task. Session-to-session alternation and ordinal timing are both based on order information, but only ordinal timing is embedded in a temporal context (i.e. lever 1 then lever 2, each day). The rats did not rely solely on a session-tosession alternation strategy. If they had, their Corr scores would have been high and their Alt scores would have been low in morning/afternoon and afternoon/morning sessions, and these values would have re6ersed magnitudes in morning/morning and afternoon/afternoon sessions. This follows because a session-to-session alternation strategy predicts food availability at the Alt lever in morning/morning and afternoon/afternoon sessions. Instead, the rats’ Alt and Corr scores changed by roughly 10% during morning/ morning and afternoon/afternoon sessions. It appears that the rats used some other type of information, in conjunction with a session-tosession alternation strategy, to track the location of food availability. When these two mechanisms predicted food availability at different locations, as they did during sessions preceded by a session at the same time of day, the result was behavioural compromise. Carr and Wilkie (1997b) found evidence that rats also use multiple types of information when tracking the location of food availability in the original daily time– place task. After their rats’ received food at their morning levers they did not expect food to be available at that location for the rest of that day, even during probe ses-

sions given at 11:45 h. This is consistent with the rats’ use of ordinal information. However, at 11:45 h the rats did not prefer their afternoon levers as strongly as they did during the baseline afternoon sessions. The rats’ preference for their afternoon levers then increased as the time of the interpolated probe session approached 15:30 h. This temporal gradient in the rats’ preference for their afternoon levers suggests that the behavioural control held by ordinal timing was moderated by the status of a second timing system. As this secondary timing system had relatively weak control over the rats’ time–place behaviour is was difficult to characterize directly. However, Carr and Wilkie (1997b) found that the discrimination performance of these rats did not decrease over a 9day constant dim LL period. This result suggests that this secondary timing system was not a light-entrained circadian clock, or interval timing from light onset or awakening each day. Consequently Carr and Wilkie suggested that the second timing system was a food-entrained circadian clock.

1 As pointed out by a reviewer, this pattern of results would also be produced by rats using a probability matching rule: The rats may have distributed their anticipatory responses in accordance with the probability that each lever would provide food (one-third to lever that provided food in the previous session, and two thirds to the alternate lever).

3.1. Materials and methods

3. Experiment 2: retraining on the standard daily time–place task In Experiment 2 we shifted the rats to our original daily time–place training protocol. We hoped to show that in response to this change in the spatiotemporal properties of food availability rats would switch strategies, and begin to track the location of food availability with ordinal timing. We reasoned that if we obtained this result, we could be confident that the rats’ performance in Experiment 1 reflected the spatiotemporal properties of food availability in the modified daily time–place task, and not some difference between these rats and those used by Carr and Wilkie (1997b).

3.1.1. Animals and apparatus The rats and apparatus were the same as those used in Experiment 1.

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3.1.2. Procedure The animals were switched to the original daily time –place training protocol for nine 7-day blocks (63 days) of morning and afternoon session days. The training procedure used was the same as that used by Carr and Wilkie (1997b). The rats were tested 5 – 7 days per week. During the last 32 days of retraining the rats skipped a single test session every 2 or 3 days. The type of session skipped (morning or afternoon) was determined according to a quasi-random schedule. A total of four morning and four afternoon sessions were skipped. These skip-session probes were administered as they were by Carr and Wilkie (1997b). When a session was skipped the rats were transported to the enrichment chamber at the usual time. The rats remained in the enrichment chamber for 45 min, and then they were transported back to their home cages. In their home cages they received their usual post-session meal plus additional food to compensate for the missed experimental session. This protocol ensured that on skip-session days the rats received roughly the same handling, enrichment experience, and amount of food, at roughly the same time as they did during retraining days during Experiment 2. During rest days the rats remained in their home cages and were fed at one of the usual post-session feeding times (i.e. at 10:30 or 16:30 h). The time of feeding on rest days was determined according to a random schedule.

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The rats appeared to adjust well to the new spatiotemporal pattern of food availability in Experiment 2, and from Block 4 onwards of retraining their morning/afternoon and afternoon/ morning overall mean Corr scores were consistently at or above 75%. Blocks 4–9 were therefore designated the retraining asymptote period. The rats’ overall mean Corr score during the retraining asymptote period was 76.79 (S.E.M.= 8.81) during morning/afternoon sessions, and 80.61 (S.E.M.= 2.77) during afternoon/morning sessions. These overall mean Corr scores were significantly greater than a conservative estimate of chance performance, [for morning/afternoon sessions one sample t(3)= 3.05, PB 0.05, for afternoon/morning sessions, one sample t(3)= 11.04, PB 0.003]. Fig. 4 presents the rats’ overall mean Corr, Alt, and Inc scores (+ S.E.M.) for the morning/afternoon and afternoon/morning sessions, for the morning/morning sessions (morning sessions that immediately followed a skipped afternoon session), and for the afternoon/afternoon sessions (afternoon sessions that immediately followed a skipped morning session) during the retraining asymptote period. As discussed above, the rats’ Corr scores in morning/afternoon and afternoon/ morning sessions during the retraining asymptote

3.2. Results The rats’ overall mean Corr scores (for blocks of seven sessions) for only the morning/afternoon and afternoon/morning sessions during the acquisition asymptote period of Experiment 12 and the morning/afternoon and afternoon/morning sessions during Experiment 2 are presented in Fig. 3. Data from the eight sessions that immediately followed a skipped session have been omitted from this analysis. 2 These data points do not match the overall mean Corr scores for the acquisition asymptote period presented in Fig. 1, as they do not include the data collected during morning/ morning and afternoon/afternoon sessions.

Fig. 3. Overall mean Corr scores (for blocks of seven sessions) for morning/afternoon and afternoon/morning sessions during the acquisition asymptote period of Experiment 1, and for morning/afternoon and afternoon/morning sessions during Experiment 2. The solid line indicates a conservative final chance level of 50%. Corr =the correct lever in that session.

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Fig. 4. Overall mean percent ( + S.E.M.) of all anticipatory responses that were on the rats’ Corr, Alt, and Inc levers during the retraining asymptote period of Experiment 2, for morning/morning, morning/afternoon, afternoon/afternoon, and afternoon/morning sessions. The solid line indicates a conservative final chance level of 50%. Corr = the correct lever in that session; Alt = the correct lever in the alternate daily session; Inc = the two levers that never provided food.

period reliably exceeded chance levels. The pattern of results obtained in morning/morning and afternoon/afternoon sessions is dramatically different from that obtained in these two types of sessions in Experiment 1. After retraining on the original daily time–place task, skipping an afternoon session had no impact on the rats’ overall mean Corr score during the following morning session. The rats’ overall mean Corr score in morning/morning sessions was now high (83.11, S.E.M.=9.93), and reliably greater than a conservative estimate of chance performance, [one sample t(3)=3.33, PB 0.05]. Additionally, there was no difference in the rats’ overall mean Corr scores during morning/morning and morning/afternoon sessions [paired t(3) = 0.46, PB 0.68]. In contrast, after retraining on the original daily time–place task skipping a morning session caused a dramatic decrease [41.96% (S.E.M.= 5.37)] in the rats’ overall mean Corr score during the following afternoon session. The decrease in this mean overall Corr score from levels in afternoon/morning sessions was statistically significant [paired t(3)= 7.81, PB 0.005]. This decrease in the rats’ overall mean Corr score in afternoon/afternoon sessions was mirrored by a significant increase in the rats’ overall mean Alt score from

levels in afternoon/morning sessions [Mean change= 40.47%, S.E.M.=7.32; t(3)= 5.53, PB 0.02]. As the morning/afternoon sessions and afternoon/afternoon sessions were both the rats’ first session of the day, the rats should have treated these sessions identically if they were solely using ordinal information to track the location of food availability. This was clearly not the case. In the afternoon sessions that followed skipped morning sessions, our rats did not prefer their morning levers as strongly as they preferred their morning levers in morning/afternoon sessions [56.28 (S.E.M.= 8.53) versus 76.79 (S.E.M.= 8.81), t(3)=6.16, PB 0.01]. Or from the other point of view, during afternoon/afternoon sessions, our rats preferred their afternoon levers more than they usually preferred their afternoon levers in morning/afternoon sessions [38.65 (S.E.M.= 5.82) versus 20.38 (S.E.M.= 7.53), t(3)= 4.15, PB 0.03].

3.3. Discussion The rats transferred well to the original daily time–place training protocol. Importantly, the shift to the original training procedure produced a marked change in the rats’ behaviour during sessions that followed skipped sessions. During Experiment 1 the rats’ overall mean Corr score decreased by approximately 10%, and their overall mean Alt score increased by roughly the same amount, during sessions which followed a skipped morning or afternoon session. We argued that this symmetrical effect of skipped morning and afternoon sessions reflected partial behavioural control by a session-to-session alternation strategy. In contrast, after the rats were retrained on the original daily time–place task we found a markedly asymmetrical effect of skipped morning and afternoon sessions. Skipped afternoon sessions now had no deleterious effect on the rats’ ability to anticipate the location of food availability during the following morning session. In contrast, during the afternoon sessions that followed skipped morning sessions the rats displayed a marked decrease in their preference for their afternoon levers, and a marked increase in their prefer-

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ence for their morning levers. This asymmetrical effect of skipped morning and afternoon sessions is identical that displayed by the rats that we tested on the original daily time – place task (Carr and Wilkie, 1997b). It appears that in response to the change in the spatiotemporal characteristics of food availability in Experiment 2, the rats switched strategies, and now used ordinal timing to track the location of food availability. They now pressed one lever during their first session of each day, and pressed a second lever during their second session of each day. In Experiment 2 we again found evidence that the rats were using multiple types of information to track the location of food availability. During the afternoon sessions that followed occasional skipped morning sessions the rats prefered their morning levers (demonstrating control by ordinal information), but this preference was lower than it was in baseline morning sessions. The ordinal position of these two types of sessions was the same (first in the day), but they differed in the time of day at which they occurred. It therefore seems likely that while ordinal timing had primary control over the rats’ time – place behaviour, a secondary mechanism did have some behavioural control.

4. General discussion After extensive training on the modified daily time –place task rats discriminated between morning and afternoon sessions. However, the rats’ discrimination performance systematically varied across the two types of morning and afternoon sessions. Analysis of this variability suggested that the rats used a session-to-session alternation strategy, in conjunction with some other unknown type of information, to track the location of food availability. During the sessions for which a session-to-session alternation strategy did not predict the location of food availability, the rats’ still prefered the correct lever, but this preference was attenuated. In Experiment 2 the rats were shifted to the original daily time – place training protocol. The results of skip-session probes given after retraining suggested that the rats had shifted strate-

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gies, and now primarily used ordinal timing to track the location of food availability. We argued that rats used another type of information, in combination with session-to-session alternation, to track the location of food availability in Experiments 1 and 2. It is possible that this second type of information was circadian phase information. This notion is consistent with the evidence that circadian timing had partial control over rats’ time–place behaviour in the original daily time–place task (Carr and Wilkie, 1997b). This hypothesis is also consistent with the conclusion reached by Mistlberger et al. (1996) that rats used circadian timing to track the location of food availability in their T-maze daily time–place task. However, circadian timing could, in principle, predict the location of food availability in all types of morning and afternoon test sessions in the modified daily time–place task. In contrast, the session-to-session alternation strategy used by the rats could only predict the location of food availability in two-thirds of test all sessions. (Recall that in the modified daily time–place task two-thirds of morning and afternoon sessions were preceded in time by a session at the alternate time of day.) Consequently, if rats can use time of day information to track daily spatiotemporal patterns of food availability, it is not clear to us why they failed to utilize it fully in our modified daily time–place task. After being retrained on the original daily time–place task the rats shifted strategies, and began to use ordinal timing to track the location of food availability. This outcome suggests that the performance of these rats on the modified daily time–place task reflected the spatiotemporal properties of food availability, and not some unknown difference between these rats and those used by Carr and Wilkie (1997b). This result also suggests that rats are able to flexibly shift timing strategies in response to subtle changes in the daily spatiotemporal characteristics of food availability. Our conclusions, and those of Mistlberger et al. (1996), are clearly discrepant. While Mistlberger et al. concluded that rats are able to use circadian phase information to track a daily spatiotemporal pattern of food availability, we have repeatedly

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failed to find robust evidence for this ability in rats. It is likely that our discrepant results reflect some difference in our testing protocols. However, there are many potentially relevant differences in our procedures, and at present we are unable to chose between them. Clearly further experimentation with a variety of tasks is necessary. At a minimum, the present data suggest that it is difficult to demonstrate rats’ ability to use time of day information to track daily spatiotemporal patterns of food availability. The stronger interpretation of these data is that they reflect a qualitative difference in rats’ and pigeons’ cognitive systems. Rats may be unable to track a daily spatiotemporal pattern of food availability with temporal information provided by a circadian clock. Rats clearly display many circadian rhythms in behavioural and biological systems. How can we reconcile this fact with rats’ failure to use circadian timing in our daily time – place task? The answer to this question may lie in the growing sentiment that there may be at least two different types of circadian timing. One type of circadian timing is thought to be based on the operation of an entrained circadian clock that engages a class of behaviors when it reaches a single criterion phase angle each oscillation. Sleep–awake cycles and daily meal anticipation in rats and pigeons are thought to be based on this type of circadian timing (Mistlberger, 1994; Mistlberger and Marchant, 1995; Rashotte and Stephan, 1996). This type of circadian timing does allow rats to anticipate two meals per day that occur at the same location in space (Bolles and Moot, 1973; Stephan, 1989a,b). This anticipation appears to be based on the food-entrained oscillator decomposing into two independent, but coupled oscillators, each entrained to a different meal. When each oscillator reaches a criterion phase angle, general anticipatory activity is engaged (Stephan, 1989a,b; Mistlberger, 1994). However, this system appears to provide go versus no-go information rather than time of day information. Consequently, it does not enable rats to associate each meal with a different time of day. A second type of circadian timing is thought to

entail discriminating, or consulting, the current phase angle of a circadian clock at any point in its 24-h oscillation. This time of day information is then used in some other decision-making process. Circadian clock consultation is thought to provide bees (Gould, 1980; Dyer and Dickinson, 1996), and pigeons (Keeton, 1974), with the time of day information necessary for sun-compass navigation (Gallistel, 1990; Dyer, 1998). Given the continuous nature of the temporal information provided by circadian clock-consultation, it would enable rats to associate each meal with a different time of day. Rats may lack this ability to use a circadian pacemaker as a continuously consultable clock. It is easy to imagine how rats could track daily patterns of food availability effectively without a circadian clock consultation ability. Meal-anticipation rhythms would ensure that rats are active during the portion(s) of the day when food has been available in the past, and ordinal timing would ensure that rats visit their feeding sites in the correct order each day. Using this circadian and ordinal information, rats would be able to visit each of their feeding sites at the correct times of day even though they are unable associate each daily feeding opportunity with a unique circadian phase. The operating characteristics of ordinal timing seem particularly well-suited to solving this problem. For example, rats show no disruption in their time–place behaviour after having skipped their previous afternoon session and being held in dim LL (Carr and Wilkie, 1997b). This suggests that rats’ ordinal timers are endogenously reset each day. This endogenous rest function would ensure that rats begin foraging each day expecting food to be available at the first location in their daily routes, irrespective of whether they completed their foraging route on the previous day. Additionally, Carr and Wilkie (1997b) found that rats display robust discrimination performance in afternoon sessions that occurred 6 h after morning sessions. This suggests that rats are able to remember their current position in their daily routes for at least 6 h. Finally, rats are able to incorporate at least three different locations into their daily routes with no appreciable decrease in their discrimination ability (Carr et al., in review).

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Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada. Jason A.R. Carr is supported by a Sir Dudley Spurling Postgraduate Scholarship.

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Rats are reluctant to use circadian timing in a daily time-place task.

On daily time-place learning tasks animals can work for food at different spatial locations during sessions at different times of the day. In previous...
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