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Age-related working memory deficits in the allocentric place discrimination task: possible involvement in cholinergic dysfunction
Neurobiology of Aging 20 (1999) 629 – 636
Age-related working memory deficits in the allocentric place discrimination task: possible involvement in cholinergic dysfunction Takefumi Kikusui*,1, Toshiyuki Tonohiro, Tsugio Kaneko Neuroscience Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan Received 6 July 1999; received in revised form 9 September 1999; accepted 5 October 1999
Abstract It is well known that learning and memory ability declines with aging. Age-related long-term changes in learning and memory ability in rats were investigated with the place navigation task and the allocentric place discrimination task (APDT) in a water maze using the same animals for each task. In a working memory place navigation task, aged animals could learn the location of the platform as well as when they were young, although strategy shifts were observed. In contrast, accuracy in the APDT significantly declined from 90% to 65% with aging. This impairment was ameliorated by an acetylcholine esterase inhibitor physostigmine at 22–23 months old. No amelioration was, however, detected in the same animals tested when they further aged to 26 –27 months old. These results suggest that the APDT performance is sensitive to age-related memory deficits and that this may be due to the cholinergic dysfunction. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Acetylcholine; Aging; Allocetric spatial cognition; Water maze; Working memory
1. Introduction In humans, it is well known that spatial learning and memory is impaired with aging [10,41,44,46]. The spatial learning and memory of aged rats are also impaired in various spatial task tests such as the Morris water maze [11,15–19]. In the Morris water maze, rats learn the location of a submerged platform in a circular pool using extramaze cues and navigate themselves onto the platform to escape from the water. This is a particularly useful tool for assessing age-related memory deficits because the motivating stimulus, escape from water, does not require food or water deprivation. Nutrient restriction in appetitive tasks, such as the radian arm maze or the T-maze, may endanger the health of aged rats. By avoiding food or water deprivation the animals can be kept in good physiological condition, especially for long-term tests to evaluate aging effects in individual animals. For these reasons, the Morris water maze * Corresponding author. Tel.: ⫹81-3-5841-5475. E-mail address: [email protected] (T. Kikusui) 1 Current address: Laboratory of Veterinary Ethology, Department of Animal Resource Sciences, Graduate School of Agriculture and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
has been used as a model for nonverbal tests of spatial cognition in animals [29,31,38,39,51]. Two types of memory, reference and working memory, have been studied in experimental animals [37]. Reference memory is trial-independent: the information once learned is relevant to every trial. Previous reports focused on agerelated reference memory deficits in water mazes [7,8,11– 13,15,16,25,39,40]. In contrast, working memory is trialdependent: the information is relevant to a limited number of trials only. The latter type of memory has a major temporal component; thus, it can represent short-term memory. It has been reported that spatial cognitive dysfunction in aged humans is severe in both working memory and shortterm memory [2,4 – 6,14,20]. Therefore, it is preferable to assess the decline of working memory ability during longterm aging by using the same animals, who have already learned a task when they were young, as a model for human senile dementia. The place navigation task (PNT) is dependent on two systems; the path integration system (i.e., how to reach the platform) and the allocentric orientation system (i.e., where the animal is and where the platform is) as previously described [33,35,36,43,49]. We previously reported that the allocentric place discrimination task (APDT), which is dependent on the allocentric orientation system only, is capa-
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ble of evaluating working memory ability both simultaneously and separately with sensory-motor and motivational systems in the same animals [23,24]. In the present study, age-related memory deficits were evaluated with the APDT and the PNT. The animals who had already learned either task, APDT or PNT, at young ages were used repeatedly for over 2 years. The decline of spatial cognitive performance with aging has been attributed to a degeneration of forebrain cholinergic projection to the hippocampus and neocortex [3,11, 21,26,27,34,45]. Physostigmine, an acetylcholine esterase inhibitor, ameliorated age-related memory deficits in the APDT at old ages. No amelioration was, however, observed in the same animals tested when they further aged.
2. Materials and methods 2.1. Subjects Male Fisher-344 rats were purchased from Charles River Japan Inc. (Yokohama, Japan). At the beginning of training, the animals were 9 to 10 weeks old. They were housed 3–5 per cage in a room with a 12:12 h light/dark cycle (light on; 0700 h–1900 h), with the environment kept at constant temperature (24 ⫾ 1°C) and humidity (45 ⫾ 5%). Food and water were provided ad lib. All experiments were performed from 0900 h to 1800 h. 2.2. Apparatus A circular water tank, 1.5 m in diameter and 0.5 m in height, was located in the center of a small room and was surrounded by numerous extramaze cues on the walls of the room. The tank was filled to a depth of 40 cm with clear water maintained at 23.5 ⫾ 1.0°C, and divided into four quadrants (N, S, E, and W) by two imaginary perpendicular lines crossing the center of the tank. The experimenter stood in the southwest corner of the room. The platforms used were round disk platforms of 12 cm in diameter. A transparent platform was used for training for both PNT and APDT, and was located 1.5 cm beneath the water surface. Two visible platforms, both made of white acrylate, were used for the APDT test and were located 0.5 cm above the water surface. These visible platforms were identical in appearance, one was fixed to the pool bottom with a plastic bar so that a rat was able to climb onto the platform from the water, whereas the other was a float connected to the pool bottom with thread and would sink when a rat tried to climb onto it. An automated color tracking system (CAT-10, Muromachi Kikai, Co., Ltd., Tokyo, Japan) recorded the position of a rat in the tank. A camera was mounted 1.5 m above the surface of the water.
2.3. Procedure Thirty-nine animals were divided into 3 groups, 13 for working memory of the PNT, 13 for reference memory of the PNT, and 13 for the APDT. The training phase for each task was started at 9 –10 weeks old for all rat groups. In the APDT test, rats were trained for the working memory of the PNT first, and then trained for the APDT as previously reported [23]. 2.3.1. PNT; reference memory 2.3.1.1. Training phase. The transparent platform was located 1.5 cm beneath the water surface in the center of the N quadrant and remained in the same location throughout all the tests. One session, consisting of 4 trials, was given each day. Rats were released into the water at one of the three quadrants not containing the platform. The sequence of start location was chosen from the three quadrants in a pseudo-random manner. A trial began by releasing a rat into the water facing the wall of the tank and ended when the rat found the platform or in 90 s, whichever came first. If the rat could not reach the platform within 90 s, the experimenter led the rat to the platform. The rat remained on the platform for 60 s and was then released into the water again from the next start location. The criterion for the acquisition of this task was that a rat could reach the platform within 2000 cm 䡠 s of search error in all trials for 3 consecutive sessions. Search error, which is the cumulative distance from the platform to the rat and reflects the spatial distribution of the rat’s search relative to the platform, was calculated according to the methods of Gallagher et al. [17]. All animals tested fulfilled the criterion within 10 sessions and were used for the aging tests. 2.3.1.2. Test phase. In the aging tests, the same procedure of place navigation task was performed as that in the training phase. One test consisting of 3 consecutive sessions was performed every 5 weeks. Search error was determined for each trial. The mean value of search error of 3 sessions for each trial was calculated. Data for 17 (4 months), 62 (14 months), and 107 (24 months) week-old representing young, middle, and old ages in the same animals were compared. 2.3.2. PNT; working memory 2.3.2.1. Training phase. The transparent platform was located 1.5 cm beneath the water surface in one of the four quadrants (N, S, E or W) and at one of three distances from the edge of the tank (20, 40 or 60 cm). The platform location remained the same throughout a session of 4 trials. Between the sessions, the platform location was varied in a pseudorandom manner. The other procedure was the same as that in the test for reference memory of the PNT. The criterion for the acquisition of this PNT was that a rat could reach the platform within 2000 cm 䡠 s of search error in the 2nd to 4th trials for 3 consecutive sessions. All animals tested fulfilled
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the criterion within 12 sessions and were used for the aging tests.
speed, still time and swimming time were measured and analyzed for age-related changes.
2.3.2.2. Test phase. The procedure, except for the platform location, was the same as that in the test for reference memory of the PNT.
2.4. Drugs
2.3.3. APDT 2.3.3.1. Training phase. The procedure for the APDT was the same as that previously reported [23]. Animals that had acquired the working memory for the PNT were used in the APDT. Two visible platforms were located in the same tank simultaneously. One test consisted of two sessions, one session per day for 2 consecutive days. One session, consisting of 6 trials, was given each day. In the first session, the solid platform was placed at the center of the N or S quadrant in a pseudo-random manner, and the float was placed in the opposite quadrant. The locations were kept in the same positions during the first session and reversed in the second session. A trial began by releasing the rat into the water facing the wall of the tank from one of the remaining two quadrants (E or W). The sequence of start location was chosen in a pseudo-random manner such that the same start location was not employed for more than 3 consecutive trials; each location was used three times during a session. The trial ended when the rat reached either the solid platform or the float or in 90 s, whichever came first. If the rat could not reach the solid platform within 90 s, the experimenter led the rat to the solid platform. With a repetition of training, all the animals turned to the solid platform and swam to it within a few seconds when they chose the float first. The rats remained on the solid platform for 45 sec and then were released into the water again. The accuracy was calculated as the ratio (%) of the number of times the solid platform was reached within 90 s from the 2nd to the 6th trials of two 6 trials sessions (10 trials in total). The 1st trial in each session was excluded from the calculation because it served as an informational trial for the location of the solid platform. Animals were used repeatedly for the APDT tests and a refresher session was inserted between the tests, which was composed of one session for testing working memory of the PNT. The criterion of the APDT was that accuracy was above 80% for 3 consecutive tests. Twenty out of the 26 animals that fulfilled the criterion were used for the aging tests. The animals that had not fulfilled the criterion received the training of place navigation task and APDT repeatedly. However, they showed stereotyped turning to right or left, i.e., they acquired the no-spatial strategy for performing the task, and never fulfilled the criterion. 2.3.3.2. Test phase. In the aging tests, the same procedure as in the training phase was performed for the APDT. Animals were used repeatedly for the APDT tests with 3–7 week intervals. The accuracy, swimming distance, swimming
Physostigmine salicylate (PHY) 0.3 mg/kg (Sigma Chemicals Co., St. Louis, MO) was dissolved in saline at a volume of 1 mL/kg and administrated intraperitoneally (i.p.) 15 min before the test of 2 consecutive sessions. Animals tested were drugged with both PHY and saline in 2 tests of each age stage. Half of the animals were administrated PHY first, and the others were saline first. The test of 22–23 month was performed after the aging test of 95 weeks, and the test of 26 –27 month was performed after the aging test of 107 weeks. The same rats, except physically damaged animals, were repeatedly used for the examination for the effects of PHY at the ages of 22–23 and 26 –27 months. 2.5. Data analysis Data analysis was performed with StatView ⫹ Graphics 4.1J (Abacus Concepts, Inc., Berkeley, CA). The significance level for all statistical tests was set at 0.05. In the PNT tests, Friedman’s test was performed for comparison of search error. Data for 17 (4 months), 62 (14 months), and 107 (24 months) week-old rats representing young, middle, and old age rats were compared by the Kruskal-Wallis test, and post hoc analysis was calculated using the Mann-Whitney U test adopted by Ryan’s procedure. For the intra-age comparison, Friedman’s test was performed and post hoc analysis was performed by the Wilcoxson’s signed-rank test adopted by Ryan’s procedure. In the APDT aging test, the Kruskal-Wallis test was performed for comparison of all parameters, and post hoc analysis was performed by the Mann-Whitteny test adopted by Ryan’s procedure for multiple comparison. In the PHY administration tests, Wilcoxson’s signed rank test was performed for the comparison of the accuracy.
3. Results 3.1. PNT: reference memory To clarify that animals can remember the task procedures and can use extramaze cues even at old ages, reference memory of the PNT was tested. All animals could find the submerged platform effectively at all ages. This indicated that reference memory was well preserved during aging, although respective search error in the 1st to the 4th trial increased with aging (Fig. 1). Intra-age comparison analysis revealed that at 17 and 62 weeks, but not at 107 weeks old, respective search error of the 2nd to the 4th trials was shorter than that in the 1st trial (Fig. 2). Respective search error in the 1st to the 4th trials varied between the 17, 62, and 107 week-old rats. Post hoc analysis revealed that there
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Fig. 1. Age-related changes in search errors for reference memory of the PNT. The search errors of the 1st to 4th trials gradually increased with aging. Data are shown as mean ⫾ SEM (n ⫽ 10 –13). 1st trial, X2 ⫽ 47, p ⬍ 0.001; 2nd trial, X2 ⫽ 38, p ⬍ 0.01; 3rd trial, X2 ⫽ 49, p ⬍ 0.0005; 4th trial, X2 ⫽ 38, p ⬍ 0.01 in Friedman’s test.
was a difference in the respective search error of the 1st to the 4th trials of those, but the differences were not agedependent (Fig. 2). 3.2. PNT: working memory In young ages, all animals were able to reach the submerged platform directly from the start points on the 2nd to 4th trials (Fig. 3). They could find the platform effectively also at old ages using information from the 1st trial. The tracing of swimming of the 2nd to the 4th trials at old ages was different from that at young ages (Fig. 3). Some animals swam at a constant distance from the wall of the pool, others swam to the center of the pool first and then found their way to the platform (Fig. 3; 62, 107 weeks, trial 2– 4). Friedman’s test revealed that search error in the 1st trial varied during the aging tests without age-dependence, and that respective search error in each of the 2nd to 4th trials increased with aging (Fig. 4). Intra-age comparison analysis revealed that respective search error of the 2nd to the 4th trials was shorter than that of the 1st trial for all ages (Fig. 5). The search error in the 2nd to the 4th, but not in the 1st trial, increased with aging when the value was compared between 17, 62, and 107 weeks old (Fig. 5). Post hoc analysis revealed that search errors of the 2nd to the 4th trials at 62 and 107 weeks were longer than the corresponding search error at 17 weeks. 3.3. APDT The accuracy was as high as about 90% at 33 weeks (7 months) and was maintained up to around 50 weeks (12 months) (Fig. 6). After 95 weeks (22 months), the accuracy was kept at about 65% (Fig. 6). Friedman’s test showed significant differences among the values during aging, and
Fig. 2. Trial-dependent changes of search errors for reference memory of the PNT in rats at age of 17 weeks (4 months), 62 weeks (14 months), and 107 weeks (24 months). Search errors of the 2nd to 4th trails at 17 and 62 weeks old, but not 107 weeks old, decreased compared to the 1st trial. Search errors of the 1st to 4th trials at 62 and 107 weeks old increased age-independently compared to those at 17 weeks old. Data are shown as mean ⫾ SEM (n ⫽ 10 –13). *p ⬍ 0.05 compared to the 1st trial within the same age [17 weeks old; X2 ⫽ 9, p ⬍ 0.05; 1st trial vs. 2nd trial, Z ⫽ ⫺2, p ⬍ 0.05; 1st trial vs. 3rd trial, Z ⫽ ⫺2, p ⬍ 0.05; 1st trial vs. 4th trial, Z ⫽ ⫺2, p ⬍ 0.05: 62 weeks old; X2 ⫽ 16, p ⬍ 0.005; 1st trial vs. 2nd trial, Z ⫽ ⫺1, p ⬎ 0.05; 1st trial vs. 3rd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 4th trial, Z ⫽ ⫺3, p ⬍ 0.05: 107 weeks old; X2 ⫽ 4, p ⫽ 0.23]. * p ⬍ 0.05 compared to the 17 weeks old [1st trial, H ⫽ 9, p ⬍ 0.05; 2nd trial, H ⫽ 9, p ⬍ 0.05; 3rd trial, H ⫽ 18, p ⬍ 0.0001; 4th trial, H ⫽ 16, p ⬍ 0.0001. 1st trial: 17 weeks vs. 62 weeks, U ⫽ 45, Z ⫽ ⫺2, p ⬎ 0.05; 17 weeks vs. 107 weeks, U ⫽ 27, Z ⫽ ⫺3, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 56, Z ⫽ ⫺1, p ⬎ 0.05. 2nd trial: 17 weeks vs. 62 weeks, U ⫽ 29, Z ⫽ ⫺3, p ⬍ 0.05; 17 weeks vs. 107 weeks, U ⫽ 38, Z ⫽ ⫺2, p ⬎ 0.05; 62 weeks vs. 107 weeks, U ⫽ 72, Z ⫽ ⫺1, p ⬎ 0.05. 3rd trial; 17 weeks vs. 62 weeks, U ⫽ 49, Z ⫽ ⫺2, p ⬎ 0.05; 17 weeks vs. 107 weeks, U ⫽ 10, Z ⫽ ⫺4, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 2, Z ⫽ ⫺4, p ⬍ 0.05. 4th trial; 17 weeks vs. 62 weeks, U ⫽ 31, Z ⫽ ⫺3, p ⬍ 0.05; 17 weeks vs. 107 weeks, U ⫽ 2, Z ⫽ ⫺4, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 35, Z ⫽ ⫺2, p ⬍ 0.05].
post hoc analysis revealed that the accuracy of animals older than 76 weeks (18 months) decreased when compared with the accuracy at 33 weeks. There was no change in swimming distance with aging except for the 1st trial (Fig. 7a). Swimming speed decreased and swimming time increased, i.e., motor deficits were observed in aged rats (Figs. 7b, d). Still time decreased with aging in the 1st and the 2nd trial, but not in the other trials (Fig. 7c). 3.4. Effect of physostigmine on the age-related working memory deficits in APDT Because age-related memory deficits were most obviously shown in the APDT among the tasks used in this study, the effect of physostigmine was investigated for this task. Physostigmine 0.3 mg/kg ameliorated the age-related reduction in accuracy at 22–23 months, but no effect of the compound was detected in those same animals at 26 –27 months (Fig. 8). Other parameters were not affected by physostigmine (data not shown).
T. Kikusui et al. / Neurobiology of Aging 20 (1999) 629 – 636
Fig. 3. Trace drawing of swimming paths of the same animal (#3) for the working memory of the PNT at the age of 17 weeks (upper), 62 weeks (middle), and 107 weeks (lower). The paths from the 2nd to 4th trials were significantly shorter than that of the first trial at all ages. The animal swam to the platform directly from the start point of the 2nd to 4th trials at 17 weeks old. At 62 and 107 weeks old, it swam to the center of the pool first, and made its way to the platform.
4. Discussion In the test for reference memory of the PNT, rats remembered the platform location at any age, as their search errors were one-tenth smaller than those of the 1st trial in the working memory of the PNT from 12 to 112 weeks old. The 1st trial of the test for working memory of the PNT is considered as a random search in the pool, since the position of the platform is renewed at every session. Sensory ability
Fig. 4. Age-related changes of search errors for the working memory of the PNT. The search errors of the 2nd to 4th trials, but not that of the 1st trial, gradually increased with aging. Data are shown as mean ⫾ SEM (n ⫽ 10 –13). Trial 1, X2 ⫽ 37, p ⬍ 0.05; second trial, X2 ⫽ 65, p ⬍ 0.0001; third trial, X2 ⫽ 63, p ⬍ 0.0001; 4th trial, X2 ⫽ 75, p ⬍ 0.0001 in Friedman’s test.
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Fig. 5. Trial-dependent changes of search errors for working memory of the PNT at the age of 17 weeks (4 months), 62 weeks (14 months), and 107 weeks (24 months). At all ages, search errors for the 2nd to 4th trials decreased similarly compared to 1st trial. Search errors of the 2nd to 4th trials, but not 1st trial, at 62 and 107 weeks old increased compared to those at 17 weeks old. Mean ⫾ SEM (n ⫽ 10 –13). # p ⬍ 0.05 compared to the 1st trial within the same age [17 weeks old; X2 ⫽ 25, p ⬍ 0.0001; 1st trial vs. 2nd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 3rd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 4th trial, Z ⫽ ⫺3, p ⬍ 0.05: 62 weeks old; X2 ⫽ 25, p ⬍ 0.0001; 1st trial vs. 2nd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 3rd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 4th trial, Z ⫽ ⫺3, p ⬍ 0.05: 107 weeks old; X2 ⫽ 14, p ⬍ 0.005; 1st trial vs. 2nd trial, Z ⫽ ⫺2, p ⬍ 0.05; 1st trial vs. 3rd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 4th trial, Z ⫽ ⫺3, p ⬍ 0.05 in Friedman’s test and post hoc Wilcoxson’s signed rank test adopted by Ryan’s procedure]. * p ⬍ 0.05 compared to the 17 weeks old [1st trial, H ⫽ 1, p ⫽ 0.60; 2nd trial, H ⫽ 10, p ⬍ 0.01; 3rd trial, H ⫽ 12, p ⬍ 0.005; 4th trial, H ⫽ 16, p ⬍ 0.0005. 2nd trial: 17 weeks vs. 62 weeks, U ⫽ 35, Z ⫽ ⫺3, p ⬍ 0.05; 17 weeks vs. 107 weeks, U ⫽ 22, Z ⫽ ⫺3, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 56, Z ⫽ ⫺1, p ⬎ 0.05. 3rd trial; 17 weeks vs. 62 weeks, U ⫽ 39, Z ⫽ ⫺2, p ⬍ 0.05; 17 weeks vs. 107 weeks, U ⫽ 12, Z ⫽ ⫺3, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 57, Z ⫽ ⫺1, p ⬎ 0.05. 4th trial; 17 weeks vs. 62 weeks, U ⫽ 20, Z ⫽ ⫺3, p ⬍ 0.05; 17 weeks vs. 107 weeks, U ⫽ 14, Z ⫽ ⫺3, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 54, Z ⫽ ⫺1, p ⬎ 0.05 in Kruskal-Wallis test, and post hoc Mann-Whitney U test adopted by Ryan’s procedure].
was so intact when they aged that rats could use the extramaze cues. Search errors of all trials varied without age-dependency. In the test for working memory of the PNT, animals swam almost directly to the platform at young ages (Fig. 3). At old ages, the animals swam along the wall of the pool, or swam to the center of the pool first and then found their way to the platform; i.e., they found the “path” to the platform (Fig. 3). Changes in the way of how to reach the platform are referred to as “strategy shifts,” and were reportedly observed in hippocampus-lesioned or aged animals in water mazes [17,33,43]. In the present study, the search error of the 2nd to 4th trials were significantly shorter than that of the first trial. This means that working memory performance of the PNT was well maintained, although the strategy shifts were observed. In the APDT, accuracy was significantly decreased with aging (Fig. 6), suggesting that the APDT performance is sensitive to age-related memory deficits. APDT perfor-
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Fig. 6. Age-related decline of accuracy in the APDT. Accuracy decreased with aging. Data are shown as mean ⫾ SEM (n ⫽ 10 –20). *p ⬍ 0.05 compared to 33 weeks old group. H ⫽ 51, p ⬍ 0.0001: 76 weeks, U ⫽ 33, Z ⫽ ⫺4, p ⬍ 0.05; 83 weeks, U ⫽ 68, Z ⫽ ⫺3, p ⬍ 0.05; 89 weeks, U ⫽ 65, Z ⫽ ⫺3, p ⬍ 0.05; 95 weeks, U ⫽ 35, Z ⫽ ⫺4, p ⬍ 0.05; 101 weeks, U ⫽ 49, Z ⫽ ⫺4, p ⬍ 0.05; 107 weeks, U ⫽ 24, Z ⫽ ⫺4, p ⬍ 0.05.
mance impairment was not due to visual sensory deficits, because aged rats could use the distal extramaze cues as shown in the PNT. We previously showed that the APDT
Fig. 8. The effect of physostigmine on age-related working memory deficits in the APDT. Physostigmine (PHY) 0.3 mg/kg ameliorated the deficits at the age of 22–23 month-old, but these effects were not observed at the age of 26 –27 month-old. Data are shown as mean ⫾ SEM (n ⫽ 5–7), * p ⬍ 0.05 compared with the saline [22–23 months old, Z ⫽ ⫺2.22, p ⬍ 0.05; 26 –27 months old, Z ⫽ 0, p ⫽ 1.0].
performance is highly and selectively dependent on the central cholinergic system [24]. In the present study, an acetylcholine esterase inhibitor physostigmine ameliorated
Fig. 7. Age-related changes of swimming distance (a), swimming speed (b), still time (c) and swimming time (d) in the APDT. Swimming speed and still time decreased, and swimming time increased with aging. Data are shown as mean ⫾ SEM (n ⫽ 10 –20). a: 1st trial, H ⫽ 26, p ⬍ 0.001; 2nd trial, H ⫽ 18, p ⫽ 0.10; 3rd trial, H ⫽ 12, p ⫽ 0.44; 4th trial, H ⫽ 11, p ⫽ 0.51; 5th trial, H ⫽ 14, p ⫽ 0.27; 6th trial, H ⫽ 12, p ⫽ 0.48. b: 1st trial, H ⫽ 18, p ⫽ 0.13; 2nd trial, H ⫽ 36, p ⬍ 0.0005; 3rd trial, H ⫽ 25, p ⬍ 0.05; 4th trial, H ⫽ 48, p ⬍ 0.0001; 5th trial, H ⫽ 45, p ⬍ 0.0001; 6th trial, H ⫽ 41, p ⬍ 0.0001. c: 1st trial, H ⫽ 17, p ⫽ 0.14; 2nd trial, H ⫽ 24, p ⬍ 0.05; 3rd trial, H ⫽ 19, p ⫽ 0.09; 4th trial, H ⫽ 30, p ⬍ 0.005; 5th trial, H ⫽ 28, p ⬍ 0.005; 6th trial, H ⫽ 32, p ⬍ 0.005. d: 1st trial, H ⫽ 48, p ⬍ 0.0001; 2nd trial, H ⫽ 27, p ⬍ 0.01; 3rd trial, H ⫽ 21, p ⫽ 0.06; 4th trial, H ⫽ 12, p ⫽ 0.47; 5th trial, H ⫽ 10, p ⫽ 0.64; 6th trial, H ⫽ 17, p ⫽ 0.14.
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the deficits in the APDT at the age of 22–23 months, suggesting that spatial memory deficits in the APDT performance are mainly attributed to the central cholinergic dysfunction as reported previously for other tasks [3,11,21, 26,27,34,45]. The ameliorating effect of physostigmine, however, disappeared with further aging (26 –27 months). Matsuoka et al. reported that the difference in efficacy of the PHY is related to changes in cholineacetyltransferase activity in the hippocampus [28]. There is the possibility that cholinergic neural degeneration becomes so severe that it can not be compensated for with cholinergic drugs. It is reported that memory deficits in human senescence, especially in Alzheimer’s disease patients, are ameliorated by cholinomimetics, and that efficacy of medication, however, diminishes with time [1]. This is the first report as far as we know that shows that cholinergic activation ameliorates age-related spatial memory deficits until a certain stage of aging in rats. The accuracy of the APDT was sensitive to age-related working memory deficits. On the other hand, in the PNT, working memory performance was persistent, as search errors of the 2nd to the 4th trials were shorter than that of the 1st trial. What is the difference between these two tasks? In the PNT, rats swim to a submerged platform using two systems, the allocentric orientation system and the path integration system, i.e., rats reach the platform according to the information of where they are and where the platform is (allocentric orientation), and how to reach the platform (path integration) [33,35,36,43,49]. If one system is impaired, rats perform the PNT using the other system; i.e., compensation takes place [17,33,43]. In the APDT, ability of the path integration is not necessary because the platforms are visible and the path from the start point to the platforms is apparently defined. So, rats reach the solid platform according to only allocentric information [23,24]. The fact that accuracy of the APDT was sensitive to agerelated working memory deficits shows that the allocentric orientation system is impaired in aged rats. If rats can not use the allocentric orientation system, they have no other way other than the path integration system to reach the submerged platform in the PNT; i.e., rats learned a “path” to the platform. Strategy shifts seen in aged animals during the PNT are thought to reflect this situation. Allocentric spatial memory is considered as a declarative memory, because it is related the memory of “which” or “where” [32,36]. To the contrary, the path integration system is dependent on procedural memory, because it is based on the animal’s movement [48,50]. These two systems are functionally different [29] and declarative memory is more severely impaired while sparing procedural memory in aged human, especially in Alzheimer’s disease patients [9,22,30,42,47]. Therefore, the APDT might be difficult for aged rats as it is in humans. In conclusion, the APDT performance is more sensitive to age-related spatial working memory deficits than the PNT performance, and the deficits may be attributed to the central cholinergic dysfunction.
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Age-related working memory deficits in the allocentric place discrimination task: possible involvement in cholinergic dysfunction Takefumi Kikusui*,1, Toshiyuki Tonohiro, Tsugio Kaneko Neuroscience Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan Received 6 July 1999; received in revised form 9 September 1999; accepted 5 October 1999
Abstract It is well known that learning and memory ability declines with aging. Age-related long-term changes in learning and memory ability in rats were investigated with the place navigation task and the allocentric place discrimination task (APDT) in a water maze using the same animals for each task. In a working memory place navigation task, aged animals could learn the location of the platform as well as when they were young, although strategy shifts were observed. In contrast, accuracy in the APDT significantly declined from 90% to 65% with aging. This impairment was ameliorated by an acetylcholine esterase inhibitor physostigmine at 22–23 months old. No amelioration was, however, detected in the same animals tested when they further aged to 26 –27 months old. These results suggest that the APDT performance is sensitive to age-related memory deficits and that this may be due to the cholinergic dysfunction. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Acetylcholine; Aging; Allocetric spatial cognition; Water maze; Working memory
1. Introduction In humans, it is well known that spatial learning and memory is impaired with aging [10,41,44,46]. The spatial learning and memory of aged rats are also impaired in various spatial task tests such as the Morris water maze [11,15–19]. In the Morris water maze, rats learn the location of a submerged platform in a circular pool using extramaze cues and navigate themselves onto the platform to escape from the water. This is a particularly useful tool for assessing age-related memory deficits because the motivating stimulus, escape from water, does not require food or water deprivation. Nutrient restriction in appetitive tasks, such as the radian arm maze or the T-maze, may endanger the health of aged rats. By avoiding food or water deprivation the animals can be kept in good physiological condition, especially for long-term tests to evaluate aging effects in individual animals. For these reasons, the Morris water maze * Corresponding author. Tel.: ⫹81-3-5841-5475. E-mail address: [email protected] (T. Kikusui) 1 Current address: Laboratory of Veterinary Ethology, Department of Animal Resource Sciences, Graduate School of Agriculture and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
has been used as a model for nonverbal tests of spatial cognition in animals [29,31,38,39,51]. Two types of memory, reference and working memory, have been studied in experimental animals [37]. Reference memory is trial-independent: the information once learned is relevant to every trial. Previous reports focused on agerelated reference memory deficits in water mazes [7,8,11– 13,15,16,25,39,40]. In contrast, working memory is trialdependent: the information is relevant to a limited number of trials only. The latter type of memory has a major temporal component; thus, it can represent short-term memory. It has been reported that spatial cognitive dysfunction in aged humans is severe in both working memory and shortterm memory [2,4 – 6,14,20]. Therefore, it is preferable to assess the decline of working memory ability during longterm aging by using the same animals, who have already learned a task when they were young, as a model for human senile dementia. The place navigation task (PNT) is dependent on two systems; the path integration system (i.e., how to reach the platform) and the allocentric orientation system (i.e., where the animal is and where the platform is) as previously described [33,35,36,43,49]. We previously reported that the allocentric place discrimination task (APDT), which is dependent on the allocentric orientation system only, is capa-
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ble of evaluating working memory ability both simultaneously and separately with sensory-motor and motivational systems in the same animals [23,24]. In the present study, age-related memory deficits were evaluated with the APDT and the PNT. The animals who had already learned either task, APDT or PNT, at young ages were used repeatedly for over 2 years. The decline of spatial cognitive performance with aging has been attributed to a degeneration of forebrain cholinergic projection to the hippocampus and neocortex [3,11, 21,26,27,34,45]. Physostigmine, an acetylcholine esterase inhibitor, ameliorated age-related memory deficits in the APDT at old ages. No amelioration was, however, observed in the same animals tested when they further aged.
2. Materials and methods 2.1. Subjects Male Fisher-344 rats were purchased from Charles River Japan Inc. (Yokohama, Japan). At the beginning of training, the animals were 9 to 10 weeks old. They were housed 3–5 per cage in a room with a 12:12 h light/dark cycle (light on; 0700 h–1900 h), with the environment kept at constant temperature (24 ⫾ 1°C) and humidity (45 ⫾ 5%). Food and water were provided ad lib. All experiments were performed from 0900 h to 1800 h. 2.2. Apparatus A circular water tank, 1.5 m in diameter and 0.5 m in height, was located in the center of a small room and was surrounded by numerous extramaze cues on the walls of the room. The tank was filled to a depth of 40 cm with clear water maintained at 23.5 ⫾ 1.0°C, and divided into four quadrants (N, S, E, and W) by two imaginary perpendicular lines crossing the center of the tank. The experimenter stood in the southwest corner of the room. The platforms used were round disk platforms of 12 cm in diameter. A transparent platform was used for training for both PNT and APDT, and was located 1.5 cm beneath the water surface. Two visible platforms, both made of white acrylate, were used for the APDT test and were located 0.5 cm above the water surface. These visible platforms were identical in appearance, one was fixed to the pool bottom with a plastic bar so that a rat was able to climb onto the platform from the water, whereas the other was a float connected to the pool bottom with thread and would sink when a rat tried to climb onto it. An automated color tracking system (CAT-10, Muromachi Kikai, Co., Ltd., Tokyo, Japan) recorded the position of a rat in the tank. A camera was mounted 1.5 m above the surface of the water.
2.3. Procedure Thirty-nine animals were divided into 3 groups, 13 for working memory of the PNT, 13 for reference memory of the PNT, and 13 for the APDT. The training phase for each task was started at 9 –10 weeks old for all rat groups. In the APDT test, rats were trained for the working memory of the PNT first, and then trained for the APDT as previously reported [23]. 2.3.1. PNT; reference memory 2.3.1.1. Training phase. The transparent platform was located 1.5 cm beneath the water surface in the center of the N quadrant and remained in the same location throughout all the tests. One session, consisting of 4 trials, was given each day. Rats were released into the water at one of the three quadrants not containing the platform. The sequence of start location was chosen from the three quadrants in a pseudo-random manner. A trial began by releasing a rat into the water facing the wall of the tank and ended when the rat found the platform or in 90 s, whichever came first. If the rat could not reach the platform within 90 s, the experimenter led the rat to the platform. The rat remained on the platform for 60 s and was then released into the water again from the next start location. The criterion for the acquisition of this task was that a rat could reach the platform within 2000 cm 䡠 s of search error in all trials for 3 consecutive sessions. Search error, which is the cumulative distance from the platform to the rat and reflects the spatial distribution of the rat’s search relative to the platform, was calculated according to the methods of Gallagher et al. [17]. All animals tested fulfilled the criterion within 10 sessions and were used for the aging tests. 2.3.1.2. Test phase. In the aging tests, the same procedure of place navigation task was performed as that in the training phase. One test consisting of 3 consecutive sessions was performed every 5 weeks. Search error was determined for each trial. The mean value of search error of 3 sessions for each trial was calculated. Data for 17 (4 months), 62 (14 months), and 107 (24 months) week-old representing young, middle, and old ages in the same animals were compared. 2.3.2. PNT; working memory 2.3.2.1. Training phase. The transparent platform was located 1.5 cm beneath the water surface in one of the four quadrants (N, S, E or W) and at one of three distances from the edge of the tank (20, 40 or 60 cm). The platform location remained the same throughout a session of 4 trials. Between the sessions, the platform location was varied in a pseudorandom manner. The other procedure was the same as that in the test for reference memory of the PNT. The criterion for the acquisition of this PNT was that a rat could reach the platform within 2000 cm 䡠 s of search error in the 2nd to 4th trials for 3 consecutive sessions. All animals tested fulfilled
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the criterion within 12 sessions and were used for the aging tests.
speed, still time and swimming time were measured and analyzed for age-related changes.
2.3.2.2. Test phase. The procedure, except for the platform location, was the same as that in the test for reference memory of the PNT.
2.4. Drugs
2.3.3. APDT 2.3.3.1. Training phase. The procedure for the APDT was the same as that previously reported [23]. Animals that had acquired the working memory for the PNT were used in the APDT. Two visible platforms were located in the same tank simultaneously. One test consisted of two sessions, one session per day for 2 consecutive days. One session, consisting of 6 trials, was given each day. In the first session, the solid platform was placed at the center of the N or S quadrant in a pseudo-random manner, and the float was placed in the opposite quadrant. The locations were kept in the same positions during the first session and reversed in the second session. A trial began by releasing the rat into the water facing the wall of the tank from one of the remaining two quadrants (E or W). The sequence of start location was chosen in a pseudo-random manner such that the same start location was not employed for more than 3 consecutive trials; each location was used three times during a session. The trial ended when the rat reached either the solid platform or the float or in 90 s, whichever came first. If the rat could not reach the solid platform within 90 s, the experimenter led the rat to the solid platform. With a repetition of training, all the animals turned to the solid platform and swam to it within a few seconds when they chose the float first. The rats remained on the solid platform for 45 sec and then were released into the water again. The accuracy was calculated as the ratio (%) of the number of times the solid platform was reached within 90 s from the 2nd to the 6th trials of two 6 trials sessions (10 trials in total). The 1st trial in each session was excluded from the calculation because it served as an informational trial for the location of the solid platform. Animals were used repeatedly for the APDT tests and a refresher session was inserted between the tests, which was composed of one session for testing working memory of the PNT. The criterion of the APDT was that accuracy was above 80% for 3 consecutive tests. Twenty out of the 26 animals that fulfilled the criterion were used for the aging tests. The animals that had not fulfilled the criterion received the training of place navigation task and APDT repeatedly. However, they showed stereotyped turning to right or left, i.e., they acquired the no-spatial strategy for performing the task, and never fulfilled the criterion. 2.3.3.2. Test phase. In the aging tests, the same procedure as in the training phase was performed for the APDT. Animals were used repeatedly for the APDT tests with 3–7 week intervals. The accuracy, swimming distance, swimming
Physostigmine salicylate (PHY) 0.3 mg/kg (Sigma Chemicals Co., St. Louis, MO) was dissolved in saline at a volume of 1 mL/kg and administrated intraperitoneally (i.p.) 15 min before the test of 2 consecutive sessions. Animals tested were drugged with both PHY and saline in 2 tests of each age stage. Half of the animals were administrated PHY first, and the others were saline first. The test of 22–23 month was performed after the aging test of 95 weeks, and the test of 26 –27 month was performed after the aging test of 107 weeks. The same rats, except physically damaged animals, were repeatedly used for the examination for the effects of PHY at the ages of 22–23 and 26 –27 months. 2.5. Data analysis Data analysis was performed with StatView ⫹ Graphics 4.1J (Abacus Concepts, Inc., Berkeley, CA). The significance level for all statistical tests was set at 0.05. In the PNT tests, Friedman’s test was performed for comparison of search error. Data for 17 (4 months), 62 (14 months), and 107 (24 months) week-old rats representing young, middle, and old age rats were compared by the Kruskal-Wallis test, and post hoc analysis was calculated using the Mann-Whitney U test adopted by Ryan’s procedure. For the intra-age comparison, Friedman’s test was performed and post hoc analysis was performed by the Wilcoxson’s signed-rank test adopted by Ryan’s procedure. In the APDT aging test, the Kruskal-Wallis test was performed for comparison of all parameters, and post hoc analysis was performed by the Mann-Whitteny test adopted by Ryan’s procedure for multiple comparison. In the PHY administration tests, Wilcoxson’s signed rank test was performed for the comparison of the accuracy.
3. Results 3.1. PNT: reference memory To clarify that animals can remember the task procedures and can use extramaze cues even at old ages, reference memory of the PNT was tested. All animals could find the submerged platform effectively at all ages. This indicated that reference memory was well preserved during aging, although respective search error in the 1st to the 4th trial increased with aging (Fig. 1). Intra-age comparison analysis revealed that at 17 and 62 weeks, but not at 107 weeks old, respective search error of the 2nd to the 4th trials was shorter than that in the 1st trial (Fig. 2). Respective search error in the 1st to the 4th trials varied between the 17, 62, and 107 week-old rats. Post hoc analysis revealed that there
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Fig. 1. Age-related changes in search errors for reference memory of the PNT. The search errors of the 1st to 4th trials gradually increased with aging. Data are shown as mean ⫾ SEM (n ⫽ 10 –13). 1st trial, X2 ⫽ 47, p ⬍ 0.001; 2nd trial, X2 ⫽ 38, p ⬍ 0.01; 3rd trial, X2 ⫽ 49, p ⬍ 0.0005; 4th trial, X2 ⫽ 38, p ⬍ 0.01 in Friedman’s test.
was a difference in the respective search error of the 1st to the 4th trials of those, but the differences were not agedependent (Fig. 2). 3.2. PNT: working memory In young ages, all animals were able to reach the submerged platform directly from the start points on the 2nd to 4th trials (Fig. 3). They could find the platform effectively also at old ages using information from the 1st trial. The tracing of swimming of the 2nd to the 4th trials at old ages was different from that at young ages (Fig. 3). Some animals swam at a constant distance from the wall of the pool, others swam to the center of the pool first and then found their way to the platform (Fig. 3; 62, 107 weeks, trial 2– 4). Friedman’s test revealed that search error in the 1st trial varied during the aging tests without age-dependence, and that respective search error in each of the 2nd to 4th trials increased with aging (Fig. 4). Intra-age comparison analysis revealed that respective search error of the 2nd to the 4th trials was shorter than that of the 1st trial for all ages (Fig. 5). The search error in the 2nd to the 4th, but not in the 1st trial, increased with aging when the value was compared between 17, 62, and 107 weeks old (Fig. 5). Post hoc analysis revealed that search errors of the 2nd to the 4th trials at 62 and 107 weeks were longer than the corresponding search error at 17 weeks. 3.3. APDT The accuracy was as high as about 90% at 33 weeks (7 months) and was maintained up to around 50 weeks (12 months) (Fig. 6). After 95 weeks (22 months), the accuracy was kept at about 65% (Fig. 6). Friedman’s test showed significant differences among the values during aging, and
Fig. 2. Trial-dependent changes of search errors for reference memory of the PNT in rats at age of 17 weeks (4 months), 62 weeks (14 months), and 107 weeks (24 months). Search errors of the 2nd to 4th trails at 17 and 62 weeks old, but not 107 weeks old, decreased compared to the 1st trial. Search errors of the 1st to 4th trials at 62 and 107 weeks old increased age-independently compared to those at 17 weeks old. Data are shown as mean ⫾ SEM (n ⫽ 10 –13). *p ⬍ 0.05 compared to the 1st trial within the same age [17 weeks old; X2 ⫽ 9, p ⬍ 0.05; 1st trial vs. 2nd trial, Z ⫽ ⫺2, p ⬍ 0.05; 1st trial vs. 3rd trial, Z ⫽ ⫺2, p ⬍ 0.05; 1st trial vs. 4th trial, Z ⫽ ⫺2, p ⬍ 0.05: 62 weeks old; X2 ⫽ 16, p ⬍ 0.005; 1st trial vs. 2nd trial, Z ⫽ ⫺1, p ⬎ 0.05; 1st trial vs. 3rd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 4th trial, Z ⫽ ⫺3, p ⬍ 0.05: 107 weeks old; X2 ⫽ 4, p ⫽ 0.23]. * p ⬍ 0.05 compared to the 17 weeks old [1st trial, H ⫽ 9, p ⬍ 0.05; 2nd trial, H ⫽ 9, p ⬍ 0.05; 3rd trial, H ⫽ 18, p ⬍ 0.0001; 4th trial, H ⫽ 16, p ⬍ 0.0001. 1st trial: 17 weeks vs. 62 weeks, U ⫽ 45, Z ⫽ ⫺2, p ⬎ 0.05; 17 weeks vs. 107 weeks, U ⫽ 27, Z ⫽ ⫺3, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 56, Z ⫽ ⫺1, p ⬎ 0.05. 2nd trial: 17 weeks vs. 62 weeks, U ⫽ 29, Z ⫽ ⫺3, p ⬍ 0.05; 17 weeks vs. 107 weeks, U ⫽ 38, Z ⫽ ⫺2, p ⬎ 0.05; 62 weeks vs. 107 weeks, U ⫽ 72, Z ⫽ ⫺1, p ⬎ 0.05. 3rd trial; 17 weeks vs. 62 weeks, U ⫽ 49, Z ⫽ ⫺2, p ⬎ 0.05; 17 weeks vs. 107 weeks, U ⫽ 10, Z ⫽ ⫺4, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 2, Z ⫽ ⫺4, p ⬍ 0.05. 4th trial; 17 weeks vs. 62 weeks, U ⫽ 31, Z ⫽ ⫺3, p ⬍ 0.05; 17 weeks vs. 107 weeks, U ⫽ 2, Z ⫽ ⫺4, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 35, Z ⫽ ⫺2, p ⬍ 0.05].
post hoc analysis revealed that the accuracy of animals older than 76 weeks (18 months) decreased when compared with the accuracy at 33 weeks. There was no change in swimming distance with aging except for the 1st trial (Fig. 7a). Swimming speed decreased and swimming time increased, i.e., motor deficits were observed in aged rats (Figs. 7b, d). Still time decreased with aging in the 1st and the 2nd trial, but not in the other trials (Fig. 7c). 3.4. Effect of physostigmine on the age-related working memory deficits in APDT Because age-related memory deficits were most obviously shown in the APDT among the tasks used in this study, the effect of physostigmine was investigated for this task. Physostigmine 0.3 mg/kg ameliorated the age-related reduction in accuracy at 22–23 months, but no effect of the compound was detected in those same animals at 26 –27 months (Fig. 8). Other parameters were not affected by physostigmine (data not shown).
T. Kikusui et al. / Neurobiology of Aging 20 (1999) 629 – 636
Fig. 3. Trace drawing of swimming paths of the same animal (#3) for the working memory of the PNT at the age of 17 weeks (upper), 62 weeks (middle), and 107 weeks (lower). The paths from the 2nd to 4th trials were significantly shorter than that of the first trial at all ages. The animal swam to the platform directly from the start point of the 2nd to 4th trials at 17 weeks old. At 62 and 107 weeks old, it swam to the center of the pool first, and made its way to the platform.
4. Discussion In the test for reference memory of the PNT, rats remembered the platform location at any age, as their search errors were one-tenth smaller than those of the 1st trial in the working memory of the PNT from 12 to 112 weeks old. The 1st trial of the test for working memory of the PNT is considered as a random search in the pool, since the position of the platform is renewed at every session. Sensory ability
Fig. 4. Age-related changes of search errors for the working memory of the PNT. The search errors of the 2nd to 4th trials, but not that of the 1st trial, gradually increased with aging. Data are shown as mean ⫾ SEM (n ⫽ 10 –13). Trial 1, X2 ⫽ 37, p ⬍ 0.05; second trial, X2 ⫽ 65, p ⬍ 0.0001; third trial, X2 ⫽ 63, p ⬍ 0.0001; 4th trial, X2 ⫽ 75, p ⬍ 0.0001 in Friedman’s test.
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Fig. 5. Trial-dependent changes of search errors for working memory of the PNT at the age of 17 weeks (4 months), 62 weeks (14 months), and 107 weeks (24 months). At all ages, search errors for the 2nd to 4th trials decreased similarly compared to 1st trial. Search errors of the 2nd to 4th trials, but not 1st trial, at 62 and 107 weeks old increased compared to those at 17 weeks old. Mean ⫾ SEM (n ⫽ 10 –13). # p ⬍ 0.05 compared to the 1st trial within the same age [17 weeks old; X2 ⫽ 25, p ⬍ 0.0001; 1st trial vs. 2nd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 3rd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 4th trial, Z ⫽ ⫺3, p ⬍ 0.05: 62 weeks old; X2 ⫽ 25, p ⬍ 0.0001; 1st trial vs. 2nd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 3rd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 4th trial, Z ⫽ ⫺3, p ⬍ 0.05: 107 weeks old; X2 ⫽ 14, p ⬍ 0.005; 1st trial vs. 2nd trial, Z ⫽ ⫺2, p ⬍ 0.05; 1st trial vs. 3rd trial, Z ⫽ ⫺3, p ⬍ 0.05; 1st trial vs. 4th trial, Z ⫽ ⫺3, p ⬍ 0.05 in Friedman’s test and post hoc Wilcoxson’s signed rank test adopted by Ryan’s procedure]. * p ⬍ 0.05 compared to the 17 weeks old [1st trial, H ⫽ 1, p ⫽ 0.60; 2nd trial, H ⫽ 10, p ⬍ 0.01; 3rd trial, H ⫽ 12, p ⬍ 0.005; 4th trial, H ⫽ 16, p ⬍ 0.0005. 2nd trial: 17 weeks vs. 62 weeks, U ⫽ 35, Z ⫽ ⫺3, p ⬍ 0.05; 17 weeks vs. 107 weeks, U ⫽ 22, Z ⫽ ⫺3, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 56, Z ⫽ ⫺1, p ⬎ 0.05. 3rd trial; 17 weeks vs. 62 weeks, U ⫽ 39, Z ⫽ ⫺2, p ⬍ 0.05; 17 weeks vs. 107 weeks, U ⫽ 12, Z ⫽ ⫺3, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 57, Z ⫽ ⫺1, p ⬎ 0.05. 4th trial; 17 weeks vs. 62 weeks, U ⫽ 20, Z ⫽ ⫺3, p ⬍ 0.05; 17 weeks vs. 107 weeks, U ⫽ 14, Z ⫽ ⫺3, p ⬍ 0.05; 62 weeks vs. 107 weeks, U ⫽ 54, Z ⫽ ⫺1, p ⬎ 0.05 in Kruskal-Wallis test, and post hoc Mann-Whitney U test adopted by Ryan’s procedure].
was so intact when they aged that rats could use the extramaze cues. Search errors of all trials varied without age-dependency. In the test for working memory of the PNT, animals swam almost directly to the platform at young ages (Fig. 3). At old ages, the animals swam along the wall of the pool, or swam to the center of the pool first and then found their way to the platform; i.e., they found the “path” to the platform (Fig. 3). Changes in the way of how to reach the platform are referred to as “strategy shifts,” and were reportedly observed in hippocampus-lesioned or aged animals in water mazes [17,33,43]. In the present study, the search error of the 2nd to 4th trials were significantly shorter than that of the first trial. This means that working memory performance of the PNT was well maintained, although the strategy shifts were observed. In the APDT, accuracy was significantly decreased with aging (Fig. 6), suggesting that the APDT performance is sensitive to age-related memory deficits. APDT perfor-
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Fig. 6. Age-related decline of accuracy in the APDT. Accuracy decreased with aging. Data are shown as mean ⫾ SEM (n ⫽ 10 –20). *p ⬍ 0.05 compared to 33 weeks old group. H ⫽ 51, p ⬍ 0.0001: 76 weeks, U ⫽ 33, Z ⫽ ⫺4, p ⬍ 0.05; 83 weeks, U ⫽ 68, Z ⫽ ⫺3, p ⬍ 0.05; 89 weeks, U ⫽ 65, Z ⫽ ⫺3, p ⬍ 0.05; 95 weeks, U ⫽ 35, Z ⫽ ⫺4, p ⬍ 0.05; 101 weeks, U ⫽ 49, Z ⫽ ⫺4, p ⬍ 0.05; 107 weeks, U ⫽ 24, Z ⫽ ⫺4, p ⬍ 0.05.
mance impairment was not due to visual sensory deficits, because aged rats could use the distal extramaze cues as shown in the PNT. We previously showed that the APDT
Fig. 8. The effect of physostigmine on age-related working memory deficits in the APDT. Physostigmine (PHY) 0.3 mg/kg ameliorated the deficits at the age of 22–23 month-old, but these effects were not observed at the age of 26 –27 month-old. Data are shown as mean ⫾ SEM (n ⫽ 5–7), * p ⬍ 0.05 compared with the saline [22–23 months old, Z ⫽ ⫺2.22, p ⬍ 0.05; 26 –27 months old, Z ⫽ 0, p ⫽ 1.0].
performance is highly and selectively dependent on the central cholinergic system [24]. In the present study, an acetylcholine esterase inhibitor physostigmine ameliorated
Fig. 7. Age-related changes of swimming distance (a), swimming speed (b), still time (c) and swimming time (d) in the APDT. Swimming speed and still time decreased, and swimming time increased with aging. Data are shown as mean ⫾ SEM (n ⫽ 10 –20). a: 1st trial, H ⫽ 26, p ⬍ 0.001; 2nd trial, H ⫽ 18, p ⫽ 0.10; 3rd trial, H ⫽ 12, p ⫽ 0.44; 4th trial, H ⫽ 11, p ⫽ 0.51; 5th trial, H ⫽ 14, p ⫽ 0.27; 6th trial, H ⫽ 12, p ⫽ 0.48. b: 1st trial, H ⫽ 18, p ⫽ 0.13; 2nd trial, H ⫽ 36, p ⬍ 0.0005; 3rd trial, H ⫽ 25, p ⬍ 0.05; 4th trial, H ⫽ 48, p ⬍ 0.0001; 5th trial, H ⫽ 45, p ⬍ 0.0001; 6th trial, H ⫽ 41, p ⬍ 0.0001. c: 1st trial, H ⫽ 17, p ⫽ 0.14; 2nd trial, H ⫽ 24, p ⬍ 0.05; 3rd trial, H ⫽ 19, p ⫽ 0.09; 4th trial, H ⫽ 30, p ⬍ 0.005; 5th trial, H ⫽ 28, p ⬍ 0.005; 6th trial, H ⫽ 32, p ⬍ 0.005. d: 1st trial, H ⫽ 48, p ⬍ 0.0001; 2nd trial, H ⫽ 27, p ⬍ 0.01; 3rd trial, H ⫽ 21, p ⫽ 0.06; 4th trial, H ⫽ 12, p ⫽ 0.47; 5th trial, H ⫽ 10, p ⫽ 0.64; 6th trial, H ⫽ 17, p ⫽ 0.14.
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the deficits in the APDT at the age of 22–23 months, suggesting that spatial memory deficits in the APDT performance are mainly attributed to the central cholinergic dysfunction as reported previously for other tasks [3,11,21, 26,27,34,45]. The ameliorating effect of physostigmine, however, disappeared with further aging (26 –27 months). Matsuoka et al. reported that the difference in efficacy of the PHY is related to changes in cholineacetyltransferase activity in the hippocampus [28]. There is the possibility that cholinergic neural degeneration becomes so severe that it can not be compensated for with cholinergic drugs. It is reported that memory deficits in human senescence, especially in Alzheimer’s disease patients, are ameliorated by cholinomimetics, and that efficacy of medication, however, diminishes with time [1]. This is the first report as far as we know that shows that cholinergic activation ameliorates age-related spatial memory deficits until a certain stage of aging in rats. The accuracy of the APDT was sensitive to age-related working memory deficits. On the other hand, in the PNT, working memory performance was persistent, as search errors of the 2nd to the 4th trials were shorter than that of the 1st trial. What is the difference between these two tasks? In the PNT, rats swim to a submerged platform using two systems, the allocentric orientation system and the path integration system, i.e., rats reach the platform according to the information of where they are and where the platform is (allocentric orientation), and how to reach the platform (path integration) [33,35,36,43,49]. If one system is impaired, rats perform the PNT using the other system; i.e., compensation takes place [17,33,43]. In the APDT, ability of the path integration is not necessary because the platforms are visible and the path from the start point to the platforms is apparently defined. So, rats reach the solid platform according to only allocentric information [23,24]. The fact that accuracy of the APDT was sensitive to agerelated working memory deficits shows that the allocentric orientation system is impaired in aged rats. If rats can not use the allocentric orientation system, they have no other way other than the path integration system to reach the submerged platform in the PNT; i.e., rats learned a “path” to the platform. Strategy shifts seen in aged animals during the PNT are thought to reflect this situation. Allocentric spatial memory is considered as a declarative memory, because it is related the memory of “which” or “where” [32,36]. To the contrary, the path integration system is dependent on procedural memory, because it is based on the animal’s movement [48,50]. These two systems are functionally different [29] and declarative memory is more severely impaired while sparing procedural memory in aged human, especially in Alzheimer’s disease patients [9,22,30,42,47]. Therefore, the APDT might be difficult for aged rats as it is in humans. In conclusion, the APDT performance is more sensitive to age-related spatial working memory deficits than the PNT performance, and the deficits may be attributed to the central cholinergic dysfunction.
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