Evidence for a paradoxical sleep window for place learning in the Morris water maze

Physiology & Behavior, Vol. 59, No. 1, 93-97, 1996 Copyright © 1995 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/96 $15.00 + .00

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Evidence for a Paradoxical Sleep Window for Place Learning in the Morris Water Maze C A R L Y L E S M I T H .1 A N D G R E G O R Y M. ROSE

*Department of Psychology, Trent University, Peterborough, Ontario, K9J 7B8 Canada and tDepartment of Pharmacology and Neuroscience Training Program, UCHSC, and Medical Research VAMC, Denver, CO Received 19 December 1994 SMITH, C. AND G. M. ROSE. Evidence for a paradoxical sleep window for place learning in the Morris water maze. PHYSIOL BEHAV 59(1) 93-97, 1996.--Sprague-Dawley rats were given 4 consecutive days of training in the hidden platform version of the Morris water maze. They were then given paradoxical sleep deprivation (PSD) for 12 h either immediately after each training session or after a 12-h delay. The former group showed impaired learning on the second day of training compared to the 12-h-delayed PSD group or a nondeprived control group. In a second experiment, three groups of rats were trained as before and then given PSD for a 4-h interval beginning either immediately after, 4 h after, or 8 h after the end of each training session. Only rats exposed to PSD during the period beginning 4 h after the end of training each day showed an acquisition deficit. In a third experiment, rats were trained in a visible platform version of the water maze and exposed to PSD for a 12-h period either beginning immediately after the last training trial each day or after a 12-h rest delay. Neither of these groups was impaired on the task compared to a non-PSD control group. These results suggest that there is a PS window for place, but not cue, learning in the Morris water maze. Paradoxical sleep Paradoxical sleep deprivation Water maze Sprague-Dawley rat

Learning

IT now seems likely that paradoxical sleep (PS), or rapid eye movement (REM) sleep, is involved in the processing of memory. The most convincing results have come from animal studies, of which there are several extensive reviews (6,12,17,22). Briefly, two lines of evidence support this idea: 1) increases in PS above normal levels have been observed following task acquisition (3,19,25,27,28); and 2) selective deprivation of PS (PSD) results in memory deficits (11,18,23,24). In rats, it has been shown that the PS necessary for memory is confined to distinct posttraining periods, which have been named PS windows (PSW). The characteristics of these PSWs vary with the strain of animal, nature of the task, and number of training trials per session (22). In rats, it has long been known that hippocampal theta rhythm is present during PS (20,31). Loss of hippocampal theta rhythm is associated with spatial memory deficits in rats (34). Further, it has been reported that hippocampal place cells, when activated by exposure to their place field during the awake state, have elevated firing activity during subsequent PS (16). Finally, electrical stimulation patterned to mimic theta rhythm is very effective in inducing long-term potentiation (LTP), a lasting increase in synaptic strength that is thought to serve as a memory-encoding device. LTP induced by such patterned stimulation has been

Memory

Place learning

demonstrated in both the hippocampus proper (5,10,21) and the dentate gyrus (7,33). Taken together, these observations suggest the possibility that endogenous plasticity mechanisms are engaged during the PSW. Implicit in this conclusion is that PSWs are involved in hippocampus-dependent memory consolidation. The goal of the present experiments was to test this idea using a task that is commonly used to examine hippocampal function in memory, the Morris water maze (13). EXPERIMENT 1 This experiment was performed to determine whether a PSW was present at any time during the 24 h following daily training in the hidden platform version of the water maze. METHOD

Animals Sprague-Dawley rats (n = 30), raised at Trent University, were 6 - 7 months of age at the time of the experiment. The

1 To whom requests for reprints should be addressed.

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animals were housed in a temperature-controlled environment with light from 0700 to 1900 h. Animals were given ad lib access to both food and water.

Apparatus The apparatus was a water tank (105 × 80 × 90 cm high). Water filled the tank to a depth of approximately 60 cm and was made opaque by adding a nontoxic powder normally used to make white watercolor paint. The platform was an inverted flowerpot with an 8.5-cm diameter base. The rat had the flowerpot base on which to rest, but it was completely hidden from view, being 2.0 cm under the surface of the water and attached to a solid frame. Further, the top of the flower pot base was covered with a beige tape to make it even more difficult to see under the water. It was impossible for the rats to stand on the bottom of the tank with their front paws resting against the platform and keep their heads above water. Thus, they were obliged to actually climb onto the platform or to continue swimming. The area of the tank was divided into four equal quadrants, indicated by markings on the outside wall of the tank that were not visible to the rat. The platform was always positioned in the center of the same quadrant. The water in the tank was fresh each day and was maintained at 23-24°C. The tank was located in a room with a number of distinguishing features visible from the tank, including a set of holding cages on one side, and a light on the other side. The experimenter always stood on a third side in the same position while the training was done. The fourth side was a blank wall.

Procedure Prior to the start of the experiment, animals were individually handled for 5 consecutive days, 10 min per day. On the first day of the task, the rats were placed in the water tank on top of the hidden platform and steadied until they stayed by themselves for at least 10 s. Then they were given 2 rain in the tank during which time they were allowed to stay on the platform or to swim around briefly before returning to the platform. If the rat left the platform and did not return to it within 2 min, the experimenter guided the animal back to the platform and let it rest there for 15 s before returning the rat to its home cage. After a 5-min rest period, the first trial was begun. Each rat was placed in one of the four quadrants of the water tank, with its head facing the wall of the tank. The time to locate the platform and the number of quadrants entered while searching for the platform were recorded. If the rat had not found the platform after 60 s, the animal was guided to it. In all cases, the rats were allowed 15 s to sit on the platform at the end of each trial. The rats were run in groups of four, so that the intertrial interval was approximately 2 min. The order of quadrants was changed each day such that subjects were never exposed to a sequence of trials that they had before. Training was always carried out between 1000 and 1200 h to control for possible circadian factors.

SMITH AND ROSE

sleep (SWS). However, the complete muscular relaxation accompanying initiation of PS caused the rats to topple into the water and wake up. After a brief swim, the animals climbed back up onto the base of the flower pot. Thus, the effect of this treatment was to selectively eliminate PS. The rats were kept on the flowerpots each day for 12 h following the last training trial and then returned to their home cages until the following day. GR13-24. These animals were placed in their home cages for 12 h after the end of the last training trial each day and then placed in the PSD apparatus for the period from 13-24 h after the last training trial. As with the animals in the 1-12-h PSD group, these rats were almost completely dry the following day. After PSD the rats were given 10 rain in their home cages before being placed in the training situation again. While in their home cages the animals were observed to groom, but never settled down to try to sleep. Control. This was a group that was never placed in the PSD apparatus. They were trained each day as were the animals in the other two groups, but then spent the entire intervening interval in their home cages. RESULTS The ANOVA for the three groups over the 4 training days revealed a significant repeated variable effect, F(3, 81) = 17.14, p < 0.001, indicating that all groups exhibited substantial reductions in their times to find the platform over the training sessions. However, there was a signL~icantgroups × days interaction, F(6, 81) = 2.86, p < 0.02, indicating that the rate of learning was different between groups. Post hoe Newman-Keuls comparisons revealed that, compared to the other two groups, GRI-12 took significantly longer to find the platform, and this impairment occurred on training day 2 ( p < 0.05). These results are shown in Fig. 1. No obvious difference in the search strategy or general swimming pattern of G R I - 1 2 was observed on any training day. The ANOVA for the number of quadrant entries for the three groups provided similar results. There was a significant effect for training days, F(3, 81)= 6.92, p < 0.001, indicating that all groups reduced the number of quadrant entries required to find WATER MAZE (HIDDEN PLATFORM) BO 50 (/1 v

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Groups This experiment was designed to see if PSD occurring sometime in the first 24 h after the training session would interfere with the learning progress of the rat. Rats were randomly assigned to one of three groups (n = 10/group). GR1-12. Immediately after training each day, the animals in this group were placed on the bases of inverted flower pots of 8.5 em diameter surrounded by water. The base of the pot extended above the level of the water by about 1 cm. There was enough room for them to rest comfortably and to engage in slow-wave

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TRAINING D A Y

FIG. 1. Mean:l:SEM time for the rats in the three groups to find the hidden platform on each of the 4 training days. Although all groups had learned the task by training day 4, GR1-12 required significantlymore time than the other groups to locate the platform on training day 2. * p < 0.05.

EVIDENCE FOR A PS WINDOW FOR PLACE LEARNING

95

WATER MAZE (HIDDEN PLATFORM)

the platform. As before, there was also significant groups × days interaction, F(6, 81) = 2.92, p < 0.02. Finally, the Newman-Keuls comparisons revealed that the G R 1 - 1 2 group made more entries into the goal quadrant on day 2 than either the GR13-24 or the control group ( p < 0.05). The data obtained for the number of quadrant entries are presented in Table 1.

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EXPERIMENT 2

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The results of Experiment 1 suggested that there was a PSW for place learning in the Morris water maze task, and that the window occurred somewhere in the first 12 h after the last training trial, particularly on day 2. Previous work with other tasks has demonstrated that PSWs tend to be about 4 h long (22). This next experiment was done to try to more precisely define the time period after training when the PSW was manifest.

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METHOD Three groups of animals were trained in the maze, with each group being given a PSD of 4-h duration at a fixed time after the end of the last training trial each day.

Groups and Procedure GR1-4. This group was given 4 h of PSD starting immedi-

GR 1 - 4 GR 5 - 8 GR 9 - 1 2

4

TRAINING DAY

FIG. 2. Mean 5: SEM time for the rats in the three groups to find the hidden platform on each of the 4 training days. In this experiment, only GR5-8 showed a significant impairment compared to the other groups. * p < 0.05.

ately after the last training trial. The rats were then returned to their home cages until the following day. GR5-8. Animals in this group were returned to their home cages immediately after the last training trial. Then, at the beginning of the fifth hour after training, they were placed in the PSD apparatus for 4 h (hours 5 - 8 after the end of training). After this, they were again returned to their home cages, where they remained until the next day's training session. GR9-12. Animals in this group were also returned to their home cages immediately after the last training trial. At the beginning of the ninth hour after training, they were placed in the PSD apparatus for 4 h (hours 9 - 1 2 after the end of training). After this, they were again returned to their home cages, where they remained until the next day.

isons revealed that G R 5 - 8 took significantly longer to find the platform on training day 2 compared to the other two groups ( p < 0.05). These results are shown in Fig. 2. No obvious difference in the search strategy or general swimming pattern of GR5-8 was observed on any training day. The ANOVA for the number of quadrant entries made before finding the platform again showed a significant reduction over training days, F(3, 63) = 14.71, p < 0.001, as well as a significant groups × days interaction, F(6, 63) = 4.37, p < 0.001. The Newman-Keuls test showed the GR5-8 group had more goal quadrant entries than either the G R 1 - 4 or GR9-12 on training day 2 ( p < 0.05). The data obtained for the number of quadrant entries are presented in Table 1.

RESULTS

EXPERIMENT 3

An ANOVA revealed that there was substantial learning in all of the groups, F(3, 6 3 ) = 10.48, p < 0.001. As observed in Experiment 1, there was also a significant group X training day interaction, F(6, 63) = 3.04, p < 0.02. Newman-Keuls comparTABLE 1 GOAL QUADRANTENTRIES Group

Day 1

GRI-12 GR13-24 Control

6.25 (2.51) 6.18 (2.19) 6.80 (2.36)

GR1-4 GR5-8 GR9-12

8.63 (4.36) 7.75 (1.91) 9.43 (4.50)

GRl-12 GR13-24 Control

6.92 (2.97) 7.20 (3.58) 4.33 (0.90)

Day 2

Experiment 1 10.60"(2.54) 7.43 (2.85) 5.83 (3.41) Experiment2 5.75 (1.67) 12.13"(2.98) 6.63 (1.87) Experiment 3 3.75 (1.26) 5.67 (1.84) 3.33 (0.75)

Day 3

Day 4

5.15 (1,28) 5.63 (3.54) 3.63 (1.39)

4.30(1.40) 6.35 (2.97) 3.48(1.36)

5.06 (2,66) 7.06 (3,55) 6.94 (1,94)

4.00(1.77) 4.31 (1.77) 4.50 (1.83)

3.58 (1.45) 3.38 (0.96) 3.13 (0.54)

3.21 (0.60) 4.17(2.18) 3.58(0.98)

Values are mean with SD in parentheses. * Group has a higher score than any of the other groups on the same training day. In addition, these scores are higher than any of the scores on the other training days for all three groups ( p < 0.05).

Modifying the water maze problem so that the goal platform is visible allows the rats to solve the task without having to use place information. To compare the performance of the rats of the previous two experiments with that in the cued situation, three groups of animal were run in a cued version of the water maze, and given PSD as in Experiment 1. METHOD

Procedure and Apparatus The water tank described above was used as before, but in this case the top of the flower pot platform extended 2 cm above the level of the water. In addition, the top of the platform was not covered with beige tape, so its dark red color was clearly visible. The training procedure was identical to that employed in Experiment 1.

Groups GR1-12. Rats in this group were given four trials per day in the maze and then were placed in the PSD apparatus for 12 h. Following this, they were returned to their home cages until the following day.

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SMITH AND ROSE

WATER MAZE (VISIBLE PLATFORM) "~5 i

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TRAINING DAY FIG. 3. Mean___SEM time for the rats in the three groups to find the visible platform on each of the 4 training days. In contrast to what was observed in Experiment 1 for hidden platform training, there were no differences between groups on any of the training days.

GR13-24. Rats in this group were allowed 12 h of rest in their home cages and were then placed in the PSD apparatus for approximately 12 h. As in Experiment 1, the rats were then returned to their home cages for 10 min prior to the training session that followed. As had been previously observed, the animals were almost completely dry except for their tails following the PSD session, and spent the interval in their home cages grooming. The rats remained active and did not settle down to sleep. Control. These rats were trained in the cued task each day but were then returned to their home cages until the following day. They were never exposed to PSD. RESULTS

Analysis of the results from the visible platform experiment revealed that there was significant improvement in time to reach the goal for all of the groups during the 4-day training regime, F(3, 4 5 ) = 17.72, p < 0.001. However, unlike what was observed in Experiments 1 and 2, there was no group × training day interaction. This result indicates that PSD did not affect the learning of either treatment group. These data are shown in Fig. 3. Analysis of quadrant entries gave the same result (data presented in Table 1). DISCUSSION The results of Experiments 1 and 2 indicate that PSD at specific intervals after training impairs acquisition of the hidden platform version of the water maze. This impairment was observed on day 2, after the rats had been exposed to their first PSD. Further PSDs did not have this same effect, because there were no differences between the control and test groups on subsequent training days. The results from Experiment 2 indicated that the most vulnerable time period for the PSD-induced acquisition deficit was at 5 - 8 h after the end of the last training trial. Experiment 3 demonstrated that when the rat was able to see the platform during training, there was no acquisition deficit.

Thus, PS appears to be involved in learning the hidden platform version, but not the visible platform version, of the water maze. The PSW observed between 5 - 8 h after training is very similar to that observed in previous studies that utilized other learning tasks. Depending upon the nature of the task, the strain of rat employed and the number of trials in a single training session, a 4-h PSW is usually somewhere between 1-12 h after the last training trial (22). However, the present results are unique in one respect, in that PSD only retarded acquisition at the beginning of training and did not interfere with the final degree of acquisition of the spatial learning task. To our knowledge, this is the first time that such a transient PSD effect has been reported. It is possible that the PSW shifted in time following the first episode of PSD. Electrophysiological recordings of the occurrence of PS would confirm or deny this hypothesis. Alternatively, it is possible that learning of the hidden platform task involves the sequential activation of several brainstructures, only the first (or early) of which require PS-related activity for normal function. It is well established that the hippocampal formation plays a critical role in place learning (1,29,30,32). In addition, it has been shown that the medial and orbital frontal, but not parietal cortical, areas are involved in learning the location of a hidden platform in the Morris water maze (9,29). Lesions of these cortical areas, as well as lesions restricted to discrete hippocampal subregions (e.g., CA3), retard place learning in the water maze (30). However, rats with these treatments show only transient learning impairments, similar to wh~t was observed in the present study. It is also known that cholinergic septal afferents to the hippocampus and neocortex are important for both the acquisition and performance of spatial learning tasks (2,4,14,15). These connections are also necessary to drive the hippocampal theta rhythm, which is a prominent feature of PS in rats. PSD, therefore, prohibits the appearance of theta rhythm. Recent work has shown that electrical stimulation of hippocampal connections patterned to mimic theta rhythm is very effective in inducing long-term potentiation (LTP), a lasting enhancement of synaptic strength that may underlie memory encoding (5,21). Thus, it is tempting to speculate that the inhibitory effect of PSD may involve, at least in part, the disruption of endogenously generated synaptic plasticity. Because activation of glutamatergic NMDA receptors is known to be necessary for most forms of hippocampal LTP, one way to test the hypothesis that an LTP-like process is occurring during PSW would be to administer selective NMDA receptor antagonists during this time. Such studies are currently in progress. It has been argued that the effects of PSD upon learning are primarily due to stress or other factors (8). In the present series of experiments, the rats received equal amounts of PSD, but at different times. That learning deficits were observed with some PSD intervals, but not others, strongly suggests that stress is not the mechanism through which PSD is acting to influence learning. Alternatively, it is theoretically possible that a "stress window" could explain the present results. There is, at present, little experimental evidence to support this idea. With respect to the present study, the recovery of performance in the late training days, despite continued application of the putative stressor (PSD), is difficult to explain simply on the basis of a stress argument. Further, if stress was the primary mechanism underlying PSD-induced learning impairments, one might expect that a greater period of stress would induce a larger learning deficit. However, the deficits observed in Experiment 1, which involved a 12-h period of PSD, and Experiment 2, which involved only a 4-h period of PSD, are equivalent. Thus, although the design of our present studies did not allow a complete separation of the effects

EVIDENCE FOR A PS WINDOW FOR PLACE LEARNING

of PSD from those of stress, a "stress window" does not seem a likely explanation for our results. In conclusion, this work indicates that selective periods of PSD retard spatial learning in the Morris water maze. In contrast, PSD had no effect upon a nonspatial version of the water maze task. Taken together, these results suggest that the hippoeampus may be an important site of PSD's action. Further, the results indicate that the processes used to encode spatial information become active during a period that begins several hours after the training session. It is possible that the activation of endogenous

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plasticity mechanisms, dependent upon endogenous EEG rhythms that accompany PS, occurs during the PSW, which is important for normal spatial learning. ACKNOWLEDGEMENTS This work was supported by a grant from the National Science and Engineering Research Council of Canada, grant AG-10755 from the National Institute on Aging, and the Veterans Affairs Medical Research Service. The authors would like to thank James Conway for his assistance with the execution of the experiments.

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