- Email: [email protected]
Long-term dietary restriction causes negative effects on cognitive functions in rats
Neurobiology of Aging 25 (2004) 325–332
Long-term dietary restriction causes negative effects on cognitive functions in rats Shuichi Yanai∗ , Yoko Okaichi, Hiroshige Okaichi Department of Psychology, Doshisha University, Kyoto 602-8580, Japan Received 4 October 2002; received in revised form 7 April 2003; accepted 28 April 2003
Abstract Long-term dietary restriction is reported to increase life span and improve age-related cognitive deficits. The present study shows that the restriction increases the life span of rats but decreases their cognitive ability. Thirty-two rats were divided into restricted and ad lib feeding groups at 2.5 months of age. The restricted rats were kept at a weight of 280 g. The restricted rats were poor in performing the Morris water maze task at 7–12 months. At 17–18 months, they were poor in performing the delayed matching-to-place task. At 24–27 months, the surviving 13 restricted and 5 ad lib rats performed the spatial discrimination task. The restricted rats were also poor in performing this task. Injection of glucose prior to the discrimination task improved their performance to the level of the ad lib rats. These results suggest that dietary restriction is beneficial for longevity but has negative effects on the performance of cognitive tasks, and that the cause of the negative effects may be a reduced availability of glucose in the food-restricted aged rats. © 2003 Elsevier Inc. All rights reserved. Keywords: Long-term dietary restriction; Aging; Spatial cognition; Memory; Glucose; Rats
1. Introduction Aging affects learning and memory in rodents [1,11,24] and in humans [7,18,30]. Compared with young rats, aged rats exhibit learning deficits in the Morris water maze task [10,11,29,33], radial arm maze task [6,38], tunnel maze task [15], and the delayed non-matching to place task in water [9,20]. It has also been reported that long-term dietary restriction causes various biological and psychological changes. As for the biological change, the beneficial effect of dietary restriction on longevity is well established. Dietary restriction increases rats’ maximum life span by about 80% if started at weaning (21 days) [21] and from 10 to 20% if started in middle age (12 or 13 months) [36]. Other beneficial effects of dietary restriction reported include: the retardation of age-related diseases such as tumors and periarteritis [4], the amelioration of immune function deterioration [35] and oxidative damage [32], and an increase in DNA repair capacities [31]. Some researchers also reported that long-term dietary restriction ameliorates age-related impairments in learning and memory. In one study [12], aged rats fed a restricted diet (30 months old), aged rats fed ad lib (22 months old), and ∗
Corresponding author. Tel.: +81-75-251-4095; fax: +81-75-251-3077. E-mail address: [email protected] (S. Yanai).
0197-4580/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0197-4580(03)00115-5
young rats fed ad lib (6 months old) were trained in a complex T-maze task. In this study, dietary restriction meant ad lib on every other day (EOD) starting at weaning. The performance of aged ad lib rats was inferior to that of young ad lib and aged restricted rats, but there was no differences between the performance of young ad lib and aged restricted rats. Therefore, the author suggested that dietary restriction induced at weaning prevents the occurrence of age-related impairments. However, other studies using the radial arm maze failed to confirm the preventive effect of long-term dietary restriction on age-related impairments. The feeding schedule in these studies was either every other day [2] or 10 g per day [5]. Thus, the reported effects of dietary restriction on age-related learning impairments are inconsistent. One reason for this inconsistency may be found in their experimental design. These studies [2,5,12] used food-motivated tasks. Therefore, the ad lib group had to be placed on a food deprivation schedule before and during training to enhance the motivational level for the task, whereas the restricted group was on their regular dietary schedule. This manipulation of dietary restriction makes it unclear whether the reward value is equivalent for the ad lib and the restricted groups. Thus, the use of a food-motivated task for examining the effect of long-term dietary restriction on learning ability seems inappropriate [19]. From this point of view, the Morris water maze [25], in which
326
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
the motivation to perform the task is independent of diet, may be more appropriate. However, inconsistency exists even among the studies using non-food-motivated tasks. A study using the Morris water maze has reported that 70% dietary restriction alleviated the age-related impairments [13]. However, in other studies using the same task, 60% dietary restriction failed to mitigate the impairments [3,19]. Putting these findings together, dietary restriction shows beneficial effects consistently on the physiological aspects including prolonged life span, but inconsistent effects concerning age-related learning impairments in both aversive and appetitive tasks. In the present study, we assessed the effects of long-term dietary restriction using several non-appetitive tasks: the Morris-type place task, the delayed matching-to-place (DMTP) task, and the spatial discrimination task in a pool. These tasks require escaping from water, and the motivation to learn the tasks is independent of diet. Moreover, all of these tasks require the spatial cognitive ability that is known to decline with age [1,6,11,22]. By using these spatial tasks, we evaluated the changes in the spatial cognitive ability throughout life and the effects of life-long dietary restriction on this ability. Although these three tasks are common in assessing spatial cognitive ability, the type of memory needed to perform these cognitive tasks is not the same. The Morris-type place task and the spatial discrimination task require reference memory for successful performance. In this context, we operationally defined reference memory as the memory about the position of the platform that does not change throughout the trials. On the other hand, working memory [28] is required to perform the DMTP task. In the DMTP task, working memory is the memory about the sample position that changes from trial to trial. The spatial cognitive abilities of the subjects were estimated from both of reference and working memory by giving them these tasks at several different ages. Evidence shows that learning and memory deficits associated with aging can be attenuated by glucose treatment [17,23,37,38]. Winocur [37] proposed a possible explanation that a lack of glucose in the brain may cause memory impairment as the amount of glucose taken up by the brain declines with age. In addition, if aged rats were placed under dietary restriction, they would suffer from a low blood glucose level, hence a low brain glucose level. In order to examine the effect of glucose on aged dietary restricted rats, we injected them with glucose and tested them on a spatial discrimination task.
2. Materials and methods 2.1. Subjects Thirty-two experimentally naive male Wistar rats obtained from Shimizu Lab Animals (Kyoto) were used. Subjects
were 2 months old when they arrived at the laboratory. Rats were individually housed in wire cages. The animal room was maintained at 24 ± 1 ◦ C with a 12:12 light/dark cycle (light onset at 07.00 h). All experiments were performed in accordance with the Doshisha University guidelines for animal experiments. 2.2. Apparatus The testing apparatus for all tasks used in this study was a vinyl-chloride circular pool that measured 147 cm in diameter and 47 cm in depth. The interior of the pool was flat black and the pool was placed on a stand 44 cm above the floor. The pool was filled with water to a depth of 12 cm [26]. The water was maintained at 23±1 ◦ C and darkened by nontoxic black carbon ink (Kaimei, Inc., Saitama). It was shallow enough even for 2-month-old rats so that their legs could reach the bottom of the pool easily [26]. There were various extra-maze stimuli, including video monitors, shelves, and a towel rack in the testing room. A video camera (Panasonic, Inc., Osaka) was mounted on the ceiling above the center of the pool, and images were recorded on a video recorder. To analyze swim distance, a Multimeter EC (Eschenbach, Inc., Germany) was used. The escape platform used for the Morris-type place task and the DMTP task was a cylinder 10 cm in diameter and 11 cm in height covered with black tape. Thus, the platform remained submerged 1 cm below the water level. In the DMTP task, the pool was divided into a start section and two choice sections by an inverted T-shaped barrier (71 cm wide and 47 cm high) made of black vinyl-chloride board (Fig. 1). The entrance to each choice section was 38 cm wide. In the information swim, a sliding door was closed in such a way that the rat could not enter the section in which the escape platform was not placed. In the test swim, the sliding door
Fig. 1. Schematic diagram illustrating the apparatus for the delayed matching-to-place task. An inverted T-shaped partition was inserted in the pool to create a start section and two choice sections. Each trial consisted of two swims: an information swim and a test swim. For the information swim, one of the choice sections that includes the platform was accessible. The correct choice section was not on the same side for more than two consecutive trials. The rat was allowed to locate the platform for 60 s. After 10 s on the platform, the rat was dried and placed in a holding cage for 60 s. For the test swim, both choice sections were accessible, and the platform was placed in the same section as it was in the information swim.
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
overlapped the barrier so that the rat could enter either section. In the spatial discrimination task, two platforms were used. The appearance of the two platforms was identical when viewed from the surface of water, but one permitted escape (true platform), and the other was inescapable (false platform). The true platform was a cylinder 10 cm in diameter and 13 cm in height covered with black tape. The uppermost 1 cm of the platform was wrapped with a white vinyl tape so that the white cue protruded 1 cm above the water level. The false platform was made of rubber and spring, and it tipped over when the subject touched it. A glucose analyzer BIOSEN 6030G (EnviteC-Wismar, Inc., Germany) was used to measure the blood glucose level. 2.3. Procedures All subjects were initially fed ad libitum. On their arrival, they were handled approximately 15 min per day for 2 days and, starting on Day 3, trained on the Morris-type place task. After completion of the place task, the subjects were divided into a restricted group (n = 16) and an ad lib group (n = 16) in such a way that the mean escape latency and the mean body weight for each group were similar. Dietary restriction was initiated when the animals were 2.5 months of age. For the restricted group, about 10 g of standard laboratory rat food (Oriental Yeast, Ltd., Tokyo) was given every day to maintain their body weight at approximately 280 g. The same food was freely available to the ad lib group. Both groups had free access to water throughout the entire experimental period. 2.3.1. Morris-type place task The pool was divided into four quadrants (NE, NW, SE, and SW) by two imaginary lines crossing the center of the pool. For each animal, the invisible platform was placed in the center of one of the quadrants and remained there for a training period of 4 days. Each rat was gently placed in the water facing the wall of the pool from one of the four starting points (N, E, S, or W) along the perimeter of the pool, and the animal was allowed to swim until it climbed onto the platform. When a subject could not reach the platform in 120 s, it was gently placed on the platform by the experimenter. In either case, the subject was left on the platform for 10 s and removed from the pool. Then it was quickly dried with a towel before being returned to the holding cage. The sequence of the starting points was chosen in a random manner with a restriction that the four different starting points were experienced every training day. Four trials were conducted each day. The intertrial interval was approximately 15 min. The first training of the place task was given when subjects were 2 months old, before the beginning of dietary restriction. Thereafter, subjects were retrained on the same task when they were at 3, 7, and 9 months of age in order to examine if dietary restriction affected the retention of spatial memory. During these retraining sessions, the position of the platform for each rat was the same as in the origi-
327
nal training. Each retraining session consisted of 4 training days as did the original session. When the subjects reached 12 months of age, the platform was moved to the opposite quadrant for each rat to assess the ability to learn a new position (Platform moved). Except for the position of the platform, the training procedure in this session was identical to the original training. Training with the moved platform continued for 5 days. 2.3.2. DMTP task Training on the DMTP task was initiated when the subjects reached 17 months of age. Since four ad lib rats and one restricted rat died by the time the DMTP task was conducted, 12 ad lib and 15 restricted rats participated in this training. Each trial consisted of two swims: an information swim and a test swim. In each swim, the subject was placed in the water at the center of the circumference of the starting section, facing the wall of the pool. In the information swim, one of the choice sections containing the invisible platform was accessible. The platform was not in the same choice section for more than two consecutive trials. When a subject climbed on the platform, it was left there for 10 s and then quickly dried and placed in a holding cage for 60 s. In the test swim, both choice sections were accessible, and the platform was placed in the same section as it had been in the preceding information swim. If the subject’s whole body, including the tail, entered either choice section, it was regarded that the subject made a choice. If the rat made an incorrect choice, that is, to enter into the choice section without the platform, the sliding door was closed and the rat was confined there for 30 s. After this period, the door was opened and the rat was allowed to find the platform in the correct choice section. When a subject climbed onto the platform, it was left there for 10 s, removed from the pool, dried quickly, and then returned to the holding cage. If a subject could not reach the platform in 60 s in the information swim or in the test swim, it was gently placed on the platform by the experimenter. Every subject was given one trial a day, and trained to a criterion of nine correct choices in 10 consecutive trials or a maximum of 40 trials. 2.3.3. Spatial discrimination task When subjects reached 24–27 months of age, they were trained on a spatial discrimination task, three trials a day for 8 days. Thirteen restricted and five ad lib rats were alive when the spatial discrimination task was performed. To estimate a possible effect of hypoglycemia in the restricted rats, they were divided into two groups, seven to the restricted-glucose group, and six to the restricted-saline group, in such a way that the groups were comparable in their average performance during the last session of the place task at 12 months. All ad lib rats were assigned to the ad lib-saline group. To the restricted-glucose group, 500 mg/kg of glucose was administered, a dose proved effective in a previous study [27]. In order to achieve the dose concentration, 12.5 g of d-(+)-glucose, anhydrous (Nacalai Tesque, Inc.,
328
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
Kyoto) was dissolved in 50 ml of physiological saline, and a volume of 2 ml/kg was intraperitoneally injected to each rat in the glucose group. The animals in the saline groups were similarly injected with physiological saline. In all cases, injection was made 30 min prior to the daily training. For each animal, the location of the true platform remained at the center of the same quadrant throughout training, but the location of the false platform alternated among the three other quadrants from one trial to the next in a pseudorandom manner. A correct response was defined as escaping onto the true platform without touching the false platform. When a subject touched the false platform before reaching the true one, it was regarded as an error, and the subject was allowed to correct itself. When a subject could not reach the true platform in 60 s, it was gently placed on the true platform by the experimenter. In either case, the subject was left on the platform for 15 s and then removed from the pool and dried quickly. The intertrial interval was approximately 10 min. Other procedures were identical to those used for the Morris-type place task. 2.3.4. Blood glucose measurement The blood glucose level in each subject was measured periodically by pricking the tail with a surgical knife and collecting 10 l of blood into a capillary tube. The rats were unanesthetized because the pricking took less than a second. After the collection procedure, the cut was quickly compressed and covered with a piece of adhesive tape. The first collection was made when each rat was 2.5 months old, before starting dietary restriction, and then at 3 months of age (after dietary restriction had begun). After that, the collection was repeated every 2 months. The number of times the blood glucose was measured depends on the life span of each rat. To minimize within-day fluctuations, blood collection procedures began at 11.00 h in all animals. 2.4. Data analysis For the analysis of swim speed and the number of trials to criterion, t tests were employed. For the number of rats achieved the criterion, χ2 test was used. All other data were analyzed by mixed-design two-way analyses of variance [16], dietary condition as a between-subject factor and blocks of training trials as a within-subject factor. Student–Newman–Keuls multiple range test was used for the post-hoc comparison.
3. Results In the 13th, 17th, 18th, 19th, 20th, 21st, 22nd, and 23rd months, one rat of the ad lib group died, and in the 16th and 24th month, two rats died. One of the two ad lib rats died at 24 months of age was able to complete the spatial discrimination task. Two restricted rats died at 18 months,
Fig. 2. Schematic diagram illustrating the measurement of correct heading direction (CHD) rate. The heading direction was decided when a rat swam one body length. If a part or whole of the platform was inside a 30◦ sector, whose apex is the rat’s head and the side lines of the sector expanding 15◦ on each side of the straight line extended from the axis, the heading direction was defined as correct.
and one at 24 months of age. Therefore, fine ad lib and 13 restricted rats survived until the end of the last task. 3.1. Place task To assess the performance of each rat, three indices were used: escape latency, swim path length, and correct heading direction (CHD) rate. Furthermore, as an auxiliary index, swim speed was calculated by dividing the path length by the latency. The heading direction was determined when a rat had swum one body length: if a part or whole of the platform was inside a 30◦ sector spanning from animal’s head, the heading direction was defined to be correct (see Fig. 2). The CHD rate for each block was recorded for each rat, and the group mean percentage was calculated. This index is considered to measure the accuracy of the search direction. The results of the place task conducted at the age of 2 months (prior to the restriction), and the results at 3, 7, 9, and 12 months of age after the restriction started, are presented in Fig. 3 (A: escape latency; B: swim path length; C: CHD rate). At 2 and 3 months of age, the two groups did not differ in any of the indices. Starting at 7 months of age, however, differences in performance emerged. At 7 months, the restricted group performed poorer than the ad lib group in all indices (latency: F(1, 30) = 4.79, P < 0.05; path length: F(1, 30) = 13.20, P < 0.01; CHD rate: F(1, 30) = 14.21, P < 0.001). At 9 months, the restricted group performed more poorly at path length and CHD rate indices (latency: F(1, 30) = 0.67, n.s.; path length: F(1, 30) = 2.88, P = 0.10; CHD rate: F(1, 30) = 7.35, P < 0.05). When the platform was moved in the test at 12 months of age, the difference in path length and CHD rate increased; the restricted rats performed more poorly than the ad lib rats (latency: F(1, 30) = 1.48, n.s.; path length: F(1, 30) = 5.62, P < 0.05; CHD rate: F(1, 30) = 24.84, P < 0.001). The main effects of blocks were significant for these three indices at all
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
329
Fig. 4. Mean percent correct for the spatial discrimination task by glucose-treated restricted group, saline-treated restricted group, and saline-treated ad lib group. The performance of the saline-treated restricted group remained at the chance level, whereas the glucose-treated restricted and ad lib groups learned the task. Vertical bars indicate S.E.M.
3.2. DMTP task (17 months) Ten out of 12 ad lib rats and 3 out of 15 restricted rats achieved the criterion (χ2 (1) = 10.71, P < 0.001). If a subject did not reach the criterion after 40 trials of training, it was given a score of 40. The mean number of trials required to reach the criterion of acquisition was 36.7 cm/sec (S.E.M.: 2.09) for the restricted group and 21.7 cm/sec (S.E.M.: 3.53) for the ad lib group. A t test showed that the mean number of trials to the criterion in the restricted group was larger than that of the ad lib group (t(25) = 3.85, P < 0.001). 3.3. Spatial discrimination task (24–27 months)
Fig. 3. The performance in the Morris-type place task conducted at 2 (prior to the restriction), 3, 7, 9, and 12 months of age: (A) escape latency; (B) swim path length; (C) CHD rate. Because of a defect in the video-recorded images, the swim path length and CHD percentage at the first block of the 7-month retraining are not shown. Vertical bars indicate S.E.M.
retraining sessions except for the CHD rate at the 9 months session. The interaction was significant only for path length at the 7 months session (F(2, 60) = 6.54, P < 0.01). Tests for simple effect showed a significant group effect at the second and fourth blocks (F(1, 90) = 27.51 and 7.86, respectively, P < 0.01). The mean swim speed throughout the retraining sessions of the place task (3, 7, 9, and 12 months sessions) was 24.8 cm/sec (S.E.M.: 1.26) for the restricted group and 20.6 cm/sec (S.E.M.: 1.13) for the ad lib group. A t test confirmed that the restricted group swam significantly faster than the ad lib group (t(30) = 2.45, P < 0.05).
The mean percent of correct responses in four six-trial blocks in the restricted-glucose group (Restricted-Glu), the restricted-saline group (Restricted-Sal), and the ad lib-saline group (Ad lib-Sal), respectively, are presented in Fig. 4. The performance of the restricted-saline group remained at the chance level, whereas the mean percentage of correct responses increased in the restricted-glucose and ad lib-saline groups. An ANOVA revealed a significant group effect (F(2, 15) = 5.05, P < 0.05) and trial-block effect (F(3, 45) = 5.36, P < 0.01). Student–Newman–Keuls multiple range test on the groups revealed a significant difference between the restricted-saline groups and the other two groups (P < 0.05). The interaction between the group and the trial blocks was not significant (F(6, 45) = 1.99, P < 0.10). 3.4. Blood glucose level and body weight The mean blood glucose level and the mean body weight up to 23 months of age are presented in Fig. 5. After the
330
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
Fig. 5. Mean blood glucose level (GLU) and mean body weight (BW) of restricted and ad lib rats. Figures under the age (month) show the number of subjects alive at each age. Vertical bars indicate S.E.M. Except for Month 2.5, there were significant differences between the ad lib and restricted groups every month in both the body weight and the blood glucose level.
beginning of dietary restriction, the blood glucose levels in the restricted rats were consistently lower than those in the ad lib rats, and were maintained at a stable low level. An ANOVA was conducted for the glucose data taken in a period from 2.5 to 11 months of age, when all subjects were alive. The analysis revealed a significant group effect (F(1, 30) = 274.87, P < 0.001), age effect (F(5, 150) = 23.94, P < 0.001), and interaction (F(5, 150) = 14.73, P < 0.001). The simple effect of group was significant at all ages (P < 0.001) except at 2.5 months of age. There was no difference in body weight between the groups at 2.5 months of age, but after the beginning of dietary restriction the mean body weight of the restricted rats remained stable at 280 g. The body weight of the ad lib rats increased until about 10 months of age, and then declined at 23 months. An ANOVA for the body weight using the data of the 2.5, 3, 5, 7, 9, and 11 months of age revealed a significant group effect (F(1, 30) = 401.91, P < 0.001), age effect (F(5, 150) = 548.70, P < 0.001), and interaction (F(5, 150) = 518.85, P < 0.001). There was a significant simple effect of age for the ad lib rats (F(5, 150) = 39.40, P < 0.001) and a group effect after 3 months (P < 0.001).
4. Discussion The effect of dietary restriction on longevity is well established. In order to examine the effect of dietary restriction and aging on the acquisition and performance of various tasks in addition to longevity, we gave our subjects spatial tasks that were independent of food motivation. The life span of the restricted rats was longer than that of the ad lib rats, but the restricted rats showed overall impairments in the tasks. In the Morris-type place task, the path length became longer and the CHD rate became lower in
the restricted rats at the age of 7 months and thereafter. In addition, the restricted rats performed poorly on the place task when the platform was moved, and also on the DMTP task and the spatial discrimination task. Altogether, these results suggest that dietary restriction has a negative effect on spatial cognition, despite a beneficial effect on longevity. It has been reported that dietary restriction either eliminates age-related learning impairments [12,13] or has no effect [2,3,5,19]. The results of the present study are not congruent with any of these findings. The discrepancy between the previous studies and our study may be explained by the differences in the tasks and the motivational level. In the studies using food-motivated tasks, the ad lib rats had to be placed under dietary restriction before the beginning of the training [2,5,12]. Because of this procedure, it is unclear if the reward value was equivalent for the restricted group and the ad lib group, making the findings from these studies unreliable. The studies that used aversive tasks were also different from ours in procedure. In a study by Gyger et al., a Morris-type pool had four panels on the rim. These panels could have served as a kind of proximal cue, making the type of the task unclear [13]. The strain of the rats used by Markowska was different from ours [19]. The extent of the dietary restriction was also different. The extent of our dietary restriction (to maintain the body weight of the restricted rats at 280 g; almost 35% of the maximum body weight of ad lib rats) was more stringent than the most of the previous researches. The body weight of the restricted rats in a study by Markowska was maintained at 60% of the ad lib counterparts [19]. These procedural differences may have caused the inconsistency in results. It has been reported that the mean life span of Wistar rats, the same strain used in the present study, is 785 days [8]. In the present study, 3 of 16 ad lib rats were alive when they were 785 days of age. Although the life span of our ad
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
lib rats seems shorter than that in the above report [8], the average life span of our restricted rats seems much longer than 785 days, because 13 out of our 16 restricted rats were alive at 785 days. Thus, our results on the beneficial effect of dietary restriction on longevity agree with the findings of other studies [21,36]. In the retraining of the place task, no difference was seen between the restricted and the ad lib rats in the escape latency except for the 7 months retraining. However, the analysis of the swim path length revealed that the restricted rats swam longer than the ad lib rats at the 7 months session and thereafter. Moreover, the calculated swim speed showed that the restricted rats swam faster in all sessions, though statistical significance was shown only at the 3 and 9 months sessions. The faster swim by the restricted rats may have compensated for their cognitive deficiency: as a result, their escape latencies nearly matched with those of the ad lib rats in most of the sessions. Considering this, the CHD rate may be a better index for assessing cognitive ability than the escape latency in the case of the present study. The mean CHD rate in the restricted group was consistently lower than that in the ad lib group at the 7-month test and thereafter. In addition to that, the restricted group swam longer and the mean CHD rate was lower than that in the ad lib group on the place task with platform moved at 12 months. Altogether, the spatial learning ability of the restricted rats is poorer than the ability of the ad lib rats in the acquisition of the new platform position as well as in the retention of the platform position. Our dietary restriction schedule was to maintain the body weight at 280 g throughout the experiment period. This schedule was more stringent than in some other studies [3,13,19], though there are some researchers who imposed similar [5] or more stringent restriction [34]. It is possible that dietary restriction may have resulted in the low body temperature in the restricted rats; we did not measure the body temperature. The low body temperature may have aggravated when the restricted rats were immersed in water. However, the water temperature was maintained at 23±1 ◦ C while the room temperature was 24 ◦ C. Moreover, the rats were dried with a towel as soon as they were out of the pool. In spite of these efforts to minimize the dissipation of body heat, we cannot exclude the possibility that the conceivable thermogenic differences resulting from dietary restriction could contribute to the impaired performance of the restricted rats. The Morris-type place task is a spatial task that requires reference memory. In this task, the platform remains in the same position throughout the trials. The restricted rats performed poorly in this task at the age of 7 months and thereafter. The spatial discrimination task also depends on reference memory since the rats have to discriminate between the true platform, which remained stationary, and the false one, which moved from one trial to another. In this task, the restricted rats without glucose injection showed very poor performance. The DMTP task is a spatial task that
331
requires working memory: the rats have to retain the memory of the platform position during the delay period. The restricted rats performed very poorly in this task. Theses results indicate that the restriction-induced deficit generalizes to more than one type of spatial memory. Altogether, our experiments suggest that the dietary restriction interferes with the spatial cognitive ability in aged rats. The blood glucose level in the restricted rats was stable and consistently lower than that in the ad lib rats. Since the blood glucose level is thought to reflect the glucose level in the brain [14], the impaired performance of the restricted rats may have been caused by the decreased amount of glucose taken up by the brain. This interpretation receives a strong support from our glucose injection data. Intraperitoneal injection at the age of 24–27 months improved the performance of the restricted group in the spatial discrimination task to the level of the ad lib group. Although many studies reveal positive effect of glucose injection [17,23,37,38], it is still unknown how administered glucose functions. We need further research on this matter.
Acknowledgments The authors are grateful to the editor and the reviewers for their helpful comments. This research was supported by the Scientific Frontier Project of Doshisha University, which was subsidized by the Ministry of Education, Culture, Sports, Science and Technology.
References [1] Barnes CA. Memory changes during normal aging: neurobiological correlates. In: Martinez J, Kesner R, editors. Neurobiology of learning and memory. San Diego, CA: Academic Press; 1998. p. 247–87. [2] Beatty WW, Clouse BA, Bierley RA. Effects of long-term restricted feeding on radial maze performance by aged rats. Neurobiol Aging 1987;8:325–7. [3] Bellush LL, Wright AM, Walker JP, Kopchick J, Colvin RA. Caloric restriction and spatial learning in old mice. Physiol Behav 1996;60:541–7. [4] Berg BN, Simms HS. Nutrition and longevity in the rat. III. Food restriction beyond 800 days. J Nutr 1961;74:23–32. [5] Bond NW, Everitt AV, Walton J. Effects of dietary restriction on radial-arm maze performance and flavor memory in aged rats. Neurobiol Aging 1989;10:27–30. [6] Caprioli A, Ghirardi O, Giuliani A, Ramacci MT, Angelucci L. Spatial learning and memory in the radial maze: a longitudinal study in rats from 4 to 25 months of age. Neurobiol Aging 1991;12:605–7. [7] Evans GW, Brennan PL, Skorpanich MA, Held D. Cognitive mapping and elderly adults: verbal and location memory for urban landmarks. J Gerontol 1984;39:452–7. [8] Everitt AV, Seedman NJ, Jones F. The effects of g and continuous food restriction, begun at ages 70 and 400 days, on collagen aging, proteinuria, incidence of pathology and longevity in the male rat. Mech Ageing Dev 1980;12:161–72. [9] Frick KM, Price DL, Koliatsos VE, Markowska AL. The effects of nerve growth factor on spatial recent memory in aged rats persist after discontinuation of treatment. J Neurosci 1997;17:2543–50.
332
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
[10] Gage FH, Dunnet SB, Björklund A. Age-related impairments in spatial memory are independent of those in sensorimotor skills. Neurobiol Aging 1989;10:347–52. [11] Gallagher M, Burwell RD. Relationship of age-related decline across several behavioral domains. Neurobiol Aging 1989;10:691–708. [12] Goodrick CL. Effects of lifelong restricted feeding on complex maze performance in rats. Age 1984;7:1–3. [13] Gyger M, Kolly D, Guigoz Y. Aging, modulation of food intake and spatial memory: a longitudinal study. Arch Gerontol Geriatr Suppl 1992;3:185–96. [14] Harada M, Sawa T, Okuda C, Matsuda T, Tanaka Y. Effects of glucose load on brain extracellular lactate concentration in conscious rats using a microdialysis technique. Horm Metab Res 1993;25:560–3. [15] Jucker M, Oettinger R, Bättig K. Age-related changes in working memory and reference memory performance and locomotor activity in the Wistar rat. Behav Neural Biol 1988;50:24–36. [16] Kirk RE. Experimental design, 2nd ed. Belmont, CA: Wadsworth; 1982. [17] Kopf SR, Baratti C. Memory modulation by post-training glucose or insulin remains evident at long retention intervals. Neurobiol Learn Memory 1996;65:189–91. [18] Maki PM, Zonderman AB, Weingartner H. Age differences in implicit memory: fragmented object identification and category exemplar generation. Psychol Aging 1999;14:284–94. [19] Markowska AL. Life-long diet restriction failed to retard cognitive aging in Fischer-344 rats. Neurobiol Aging 1999;30:177–89. [20] Markowska AL, Price D, Koliatsos VE. Selective effects of nerve growth factor on spatial recent memory assessed by a delayed nonmatching-to-position task in the water maze. J Neurosci 1996;16:3541–8. [21] McCay CM, Crowell F, Maynard LA. The effect of retarded growth upon the length of life span and upon the ultimate body size. J Nutr 1935;18:63–79. [22] Means LW, Kennard KJP. Working memory and the aged rat: deficient two-choice win-stay water-escape acquisition and retention. Physiol Behav 1991;49:301–7. [23] Messier C, Destrade C. Improvement of memory for an operant response by post-training glucose in mice. Behav Brain Res 1988;31:185–91. [24] Mizumori SJY, Kalyani A. Age and experience-dependent representational reorganization during spatial learning. Neurobiol Aging 1997;18:651–9.
[25] Morris RGM. Spatial localization does not require the presence of local cues. Learn Motiv 1981;12:239–60. [26] Okaichi H. Effects of dorsal–striatum lesions and fimbria–fornix lesions on the problem-solving strategies of rats in a shallow water maze. Cognit Affect Behav Neurosci 2001;1:229–38. [27] Okaichi Y, Okaichi H. Effects of glucose on scopolamine-induced learning deficits in rats performing the Morris water maze task. Neurobiol Learn Memory 2000;74:65–79. [28] Olton DS, Becker JT, Handelmann GE. Hippocampus, space, and memory. Behav Brain Sci 1979;2:313–65. [29] Rapp PR, Rosenberg RA, Gallagher M. An evaluation of spatial information processing in aged rats. Behav Neurosci 1987;101: 3–12. [30] Sharps MJ, Gollin ES. Memory for object locations in young and elderly adults. J Gerontl 1987;42:336–41. [31] Sohal RS, Agarwal S, Candas M, Forster MJ, Lal H. Effect of age and caloric restriction on DNA oxidative damage in different tissues of C57BL/6 mice. Mech Ageing Dev 1994;76: 215–24. [32] Sohal RS, Ku HH, Agarwal S, Forster MJ, Lal H. Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech Ageing Dev 1994;74:121–33. [33] van der Staay FJ. Shift in the performance of 24-month-old Wistar rats in the Morris water escape task: a comparison across 36 experiments. Behav Brain Res 1997;87:213–22. [34] Wada H, Hosokawa T, Saito K. Single toluene exposure and changes of response latency in shock avoidance performance. Neurotoxicol Teratol 1989;11:265–72. [35] Walford RL, Liu RK, Gerbase-Dekima M, Mathies M, Smith GS. Long-term dietary restriction and immune function in mice: response to sheep red blood cells and to mitogenic agent. Mech Ageing Dev 1973;2:447–54. [36] Weindruch R, Walford RL. Dietary restriction in mice beginning at 1 year of age: effects on life-span and spontaneous cancer incidence. Science 1982;215:1415–8. [37] Winocur G. Glucose-enhanced performance by aged rats on a test of conditional discrimination learning. Psycobiology 1995;23: 270–6. [38] Winocur G, Gagnon S. Glucose treatment attenuate spatial learning and memory deficits of aged rats on tests of hippocampal function. Neurobiol Aging 1998;19:233–41.
Long-term dietary restriction causes negative effects on cognitive functions in rats Shuichi Yanai∗ , Yoko Okaichi, Hiroshige Okaichi Department of Psychology, Doshisha University, Kyoto 602-8580, Japan Received 4 October 2002; received in revised form 7 April 2003; accepted 28 April 2003
Abstract Long-term dietary restriction is reported to increase life span and improve age-related cognitive deficits. The present study shows that the restriction increases the life span of rats but decreases their cognitive ability. Thirty-two rats were divided into restricted and ad lib feeding groups at 2.5 months of age. The restricted rats were kept at a weight of 280 g. The restricted rats were poor in performing the Morris water maze task at 7–12 months. At 17–18 months, they were poor in performing the delayed matching-to-place task. At 24–27 months, the surviving 13 restricted and 5 ad lib rats performed the spatial discrimination task. The restricted rats were also poor in performing this task. Injection of glucose prior to the discrimination task improved their performance to the level of the ad lib rats. These results suggest that dietary restriction is beneficial for longevity but has negative effects on the performance of cognitive tasks, and that the cause of the negative effects may be a reduced availability of glucose in the food-restricted aged rats. © 2003 Elsevier Inc. All rights reserved. Keywords: Long-term dietary restriction; Aging; Spatial cognition; Memory; Glucose; Rats
1. Introduction Aging affects learning and memory in rodents [1,11,24] and in humans [7,18,30]. Compared with young rats, aged rats exhibit learning deficits in the Morris water maze task [10,11,29,33], radial arm maze task [6,38], tunnel maze task [15], and the delayed non-matching to place task in water [9,20]. It has also been reported that long-term dietary restriction causes various biological and psychological changes. As for the biological change, the beneficial effect of dietary restriction on longevity is well established. Dietary restriction increases rats’ maximum life span by about 80% if started at weaning (21 days) [21] and from 10 to 20% if started in middle age (12 or 13 months) [36]. Other beneficial effects of dietary restriction reported include: the retardation of age-related diseases such as tumors and periarteritis [4], the amelioration of immune function deterioration [35] and oxidative damage [32], and an increase in DNA repair capacities [31]. Some researchers also reported that long-term dietary restriction ameliorates age-related impairments in learning and memory. In one study [12], aged rats fed a restricted diet (30 months old), aged rats fed ad lib (22 months old), and ∗
Corresponding author. Tel.: +81-75-251-4095; fax: +81-75-251-3077. E-mail address: [email protected] (S. Yanai).
0197-4580/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0197-4580(03)00115-5
young rats fed ad lib (6 months old) were trained in a complex T-maze task. In this study, dietary restriction meant ad lib on every other day (EOD) starting at weaning. The performance of aged ad lib rats was inferior to that of young ad lib and aged restricted rats, but there was no differences between the performance of young ad lib and aged restricted rats. Therefore, the author suggested that dietary restriction induced at weaning prevents the occurrence of age-related impairments. However, other studies using the radial arm maze failed to confirm the preventive effect of long-term dietary restriction on age-related impairments. The feeding schedule in these studies was either every other day [2] or 10 g per day [5]. Thus, the reported effects of dietary restriction on age-related learning impairments are inconsistent. One reason for this inconsistency may be found in their experimental design. These studies [2,5,12] used food-motivated tasks. Therefore, the ad lib group had to be placed on a food deprivation schedule before and during training to enhance the motivational level for the task, whereas the restricted group was on their regular dietary schedule. This manipulation of dietary restriction makes it unclear whether the reward value is equivalent for the ad lib and the restricted groups. Thus, the use of a food-motivated task for examining the effect of long-term dietary restriction on learning ability seems inappropriate [19]. From this point of view, the Morris water maze [25], in which
326
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
the motivation to perform the task is independent of diet, may be more appropriate. However, inconsistency exists even among the studies using non-food-motivated tasks. A study using the Morris water maze has reported that 70% dietary restriction alleviated the age-related impairments [13]. However, in other studies using the same task, 60% dietary restriction failed to mitigate the impairments [3,19]. Putting these findings together, dietary restriction shows beneficial effects consistently on the physiological aspects including prolonged life span, but inconsistent effects concerning age-related learning impairments in both aversive and appetitive tasks. In the present study, we assessed the effects of long-term dietary restriction using several non-appetitive tasks: the Morris-type place task, the delayed matching-to-place (DMTP) task, and the spatial discrimination task in a pool. These tasks require escaping from water, and the motivation to learn the tasks is independent of diet. Moreover, all of these tasks require the spatial cognitive ability that is known to decline with age [1,6,11,22]. By using these spatial tasks, we evaluated the changes in the spatial cognitive ability throughout life and the effects of life-long dietary restriction on this ability. Although these three tasks are common in assessing spatial cognitive ability, the type of memory needed to perform these cognitive tasks is not the same. The Morris-type place task and the spatial discrimination task require reference memory for successful performance. In this context, we operationally defined reference memory as the memory about the position of the platform that does not change throughout the trials. On the other hand, working memory [28] is required to perform the DMTP task. In the DMTP task, working memory is the memory about the sample position that changes from trial to trial. The spatial cognitive abilities of the subjects were estimated from both of reference and working memory by giving them these tasks at several different ages. Evidence shows that learning and memory deficits associated with aging can be attenuated by glucose treatment [17,23,37,38]. Winocur [37] proposed a possible explanation that a lack of glucose in the brain may cause memory impairment as the amount of glucose taken up by the brain declines with age. In addition, if aged rats were placed under dietary restriction, they would suffer from a low blood glucose level, hence a low brain glucose level. In order to examine the effect of glucose on aged dietary restricted rats, we injected them with glucose and tested them on a spatial discrimination task.
2. Materials and methods 2.1. Subjects Thirty-two experimentally naive male Wistar rats obtained from Shimizu Lab Animals (Kyoto) were used. Subjects
were 2 months old when they arrived at the laboratory. Rats were individually housed in wire cages. The animal room was maintained at 24 ± 1 ◦ C with a 12:12 light/dark cycle (light onset at 07.00 h). All experiments were performed in accordance with the Doshisha University guidelines for animal experiments. 2.2. Apparatus The testing apparatus for all tasks used in this study was a vinyl-chloride circular pool that measured 147 cm in diameter and 47 cm in depth. The interior of the pool was flat black and the pool was placed on a stand 44 cm above the floor. The pool was filled with water to a depth of 12 cm [26]. The water was maintained at 23±1 ◦ C and darkened by nontoxic black carbon ink (Kaimei, Inc., Saitama). It was shallow enough even for 2-month-old rats so that their legs could reach the bottom of the pool easily [26]. There were various extra-maze stimuli, including video monitors, shelves, and a towel rack in the testing room. A video camera (Panasonic, Inc., Osaka) was mounted on the ceiling above the center of the pool, and images were recorded on a video recorder. To analyze swim distance, a Multimeter EC (Eschenbach, Inc., Germany) was used. The escape platform used for the Morris-type place task and the DMTP task was a cylinder 10 cm in diameter and 11 cm in height covered with black tape. Thus, the platform remained submerged 1 cm below the water level. In the DMTP task, the pool was divided into a start section and two choice sections by an inverted T-shaped barrier (71 cm wide and 47 cm high) made of black vinyl-chloride board (Fig. 1). The entrance to each choice section was 38 cm wide. In the information swim, a sliding door was closed in such a way that the rat could not enter the section in which the escape platform was not placed. In the test swim, the sliding door
Fig. 1. Schematic diagram illustrating the apparatus for the delayed matching-to-place task. An inverted T-shaped partition was inserted in the pool to create a start section and two choice sections. Each trial consisted of two swims: an information swim and a test swim. For the information swim, one of the choice sections that includes the platform was accessible. The correct choice section was not on the same side for more than two consecutive trials. The rat was allowed to locate the platform for 60 s. After 10 s on the platform, the rat was dried and placed in a holding cage for 60 s. For the test swim, both choice sections were accessible, and the platform was placed in the same section as it was in the information swim.
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
overlapped the barrier so that the rat could enter either section. In the spatial discrimination task, two platforms were used. The appearance of the two platforms was identical when viewed from the surface of water, but one permitted escape (true platform), and the other was inescapable (false platform). The true platform was a cylinder 10 cm in diameter and 13 cm in height covered with black tape. The uppermost 1 cm of the platform was wrapped with a white vinyl tape so that the white cue protruded 1 cm above the water level. The false platform was made of rubber and spring, and it tipped over when the subject touched it. A glucose analyzer BIOSEN 6030G (EnviteC-Wismar, Inc., Germany) was used to measure the blood glucose level. 2.3. Procedures All subjects were initially fed ad libitum. On their arrival, they were handled approximately 15 min per day for 2 days and, starting on Day 3, trained on the Morris-type place task. After completion of the place task, the subjects were divided into a restricted group (n = 16) and an ad lib group (n = 16) in such a way that the mean escape latency and the mean body weight for each group were similar. Dietary restriction was initiated when the animals were 2.5 months of age. For the restricted group, about 10 g of standard laboratory rat food (Oriental Yeast, Ltd., Tokyo) was given every day to maintain their body weight at approximately 280 g. The same food was freely available to the ad lib group. Both groups had free access to water throughout the entire experimental period. 2.3.1. Morris-type place task The pool was divided into four quadrants (NE, NW, SE, and SW) by two imaginary lines crossing the center of the pool. For each animal, the invisible platform was placed in the center of one of the quadrants and remained there for a training period of 4 days. Each rat was gently placed in the water facing the wall of the pool from one of the four starting points (N, E, S, or W) along the perimeter of the pool, and the animal was allowed to swim until it climbed onto the platform. When a subject could not reach the platform in 120 s, it was gently placed on the platform by the experimenter. In either case, the subject was left on the platform for 10 s and removed from the pool. Then it was quickly dried with a towel before being returned to the holding cage. The sequence of the starting points was chosen in a random manner with a restriction that the four different starting points were experienced every training day. Four trials were conducted each day. The intertrial interval was approximately 15 min. The first training of the place task was given when subjects were 2 months old, before the beginning of dietary restriction. Thereafter, subjects were retrained on the same task when they were at 3, 7, and 9 months of age in order to examine if dietary restriction affected the retention of spatial memory. During these retraining sessions, the position of the platform for each rat was the same as in the origi-
327
nal training. Each retraining session consisted of 4 training days as did the original session. When the subjects reached 12 months of age, the platform was moved to the opposite quadrant for each rat to assess the ability to learn a new position (Platform moved). Except for the position of the platform, the training procedure in this session was identical to the original training. Training with the moved platform continued for 5 days. 2.3.2. DMTP task Training on the DMTP task was initiated when the subjects reached 17 months of age. Since four ad lib rats and one restricted rat died by the time the DMTP task was conducted, 12 ad lib and 15 restricted rats participated in this training. Each trial consisted of two swims: an information swim and a test swim. In each swim, the subject was placed in the water at the center of the circumference of the starting section, facing the wall of the pool. In the information swim, one of the choice sections containing the invisible platform was accessible. The platform was not in the same choice section for more than two consecutive trials. When a subject climbed on the platform, it was left there for 10 s and then quickly dried and placed in a holding cage for 60 s. In the test swim, both choice sections were accessible, and the platform was placed in the same section as it had been in the preceding information swim. If the subject’s whole body, including the tail, entered either choice section, it was regarded that the subject made a choice. If the rat made an incorrect choice, that is, to enter into the choice section without the platform, the sliding door was closed and the rat was confined there for 30 s. After this period, the door was opened and the rat was allowed to find the platform in the correct choice section. When a subject climbed onto the platform, it was left there for 10 s, removed from the pool, dried quickly, and then returned to the holding cage. If a subject could not reach the platform in 60 s in the information swim or in the test swim, it was gently placed on the platform by the experimenter. Every subject was given one trial a day, and trained to a criterion of nine correct choices in 10 consecutive trials or a maximum of 40 trials. 2.3.3. Spatial discrimination task When subjects reached 24–27 months of age, they were trained on a spatial discrimination task, three trials a day for 8 days. Thirteen restricted and five ad lib rats were alive when the spatial discrimination task was performed. To estimate a possible effect of hypoglycemia in the restricted rats, they were divided into two groups, seven to the restricted-glucose group, and six to the restricted-saline group, in such a way that the groups were comparable in their average performance during the last session of the place task at 12 months. All ad lib rats were assigned to the ad lib-saline group. To the restricted-glucose group, 500 mg/kg of glucose was administered, a dose proved effective in a previous study [27]. In order to achieve the dose concentration, 12.5 g of d-(+)-glucose, anhydrous (Nacalai Tesque, Inc.,
328
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
Kyoto) was dissolved in 50 ml of physiological saline, and a volume of 2 ml/kg was intraperitoneally injected to each rat in the glucose group. The animals in the saline groups were similarly injected with physiological saline. In all cases, injection was made 30 min prior to the daily training. For each animal, the location of the true platform remained at the center of the same quadrant throughout training, but the location of the false platform alternated among the three other quadrants from one trial to the next in a pseudorandom manner. A correct response was defined as escaping onto the true platform without touching the false platform. When a subject touched the false platform before reaching the true one, it was regarded as an error, and the subject was allowed to correct itself. When a subject could not reach the true platform in 60 s, it was gently placed on the true platform by the experimenter. In either case, the subject was left on the platform for 15 s and then removed from the pool and dried quickly. The intertrial interval was approximately 10 min. Other procedures were identical to those used for the Morris-type place task. 2.3.4. Blood glucose measurement The blood glucose level in each subject was measured periodically by pricking the tail with a surgical knife and collecting 10 l of blood into a capillary tube. The rats were unanesthetized because the pricking took less than a second. After the collection procedure, the cut was quickly compressed and covered with a piece of adhesive tape. The first collection was made when each rat was 2.5 months old, before starting dietary restriction, and then at 3 months of age (after dietary restriction had begun). After that, the collection was repeated every 2 months. The number of times the blood glucose was measured depends on the life span of each rat. To minimize within-day fluctuations, blood collection procedures began at 11.00 h in all animals. 2.4. Data analysis For the analysis of swim speed and the number of trials to criterion, t tests were employed. For the number of rats achieved the criterion, χ2 test was used. All other data were analyzed by mixed-design two-way analyses of variance [16], dietary condition as a between-subject factor and blocks of training trials as a within-subject factor. Student–Newman–Keuls multiple range test was used for the post-hoc comparison.
3. Results In the 13th, 17th, 18th, 19th, 20th, 21st, 22nd, and 23rd months, one rat of the ad lib group died, and in the 16th and 24th month, two rats died. One of the two ad lib rats died at 24 months of age was able to complete the spatial discrimination task. Two restricted rats died at 18 months,
Fig. 2. Schematic diagram illustrating the measurement of correct heading direction (CHD) rate. The heading direction was decided when a rat swam one body length. If a part or whole of the platform was inside a 30◦ sector, whose apex is the rat’s head and the side lines of the sector expanding 15◦ on each side of the straight line extended from the axis, the heading direction was defined as correct.
and one at 24 months of age. Therefore, fine ad lib and 13 restricted rats survived until the end of the last task. 3.1. Place task To assess the performance of each rat, three indices were used: escape latency, swim path length, and correct heading direction (CHD) rate. Furthermore, as an auxiliary index, swim speed was calculated by dividing the path length by the latency. The heading direction was determined when a rat had swum one body length: if a part or whole of the platform was inside a 30◦ sector spanning from animal’s head, the heading direction was defined to be correct (see Fig. 2). The CHD rate for each block was recorded for each rat, and the group mean percentage was calculated. This index is considered to measure the accuracy of the search direction. The results of the place task conducted at the age of 2 months (prior to the restriction), and the results at 3, 7, 9, and 12 months of age after the restriction started, are presented in Fig. 3 (A: escape latency; B: swim path length; C: CHD rate). At 2 and 3 months of age, the two groups did not differ in any of the indices. Starting at 7 months of age, however, differences in performance emerged. At 7 months, the restricted group performed poorer than the ad lib group in all indices (latency: F(1, 30) = 4.79, P < 0.05; path length: F(1, 30) = 13.20, P < 0.01; CHD rate: F(1, 30) = 14.21, P < 0.001). At 9 months, the restricted group performed more poorly at path length and CHD rate indices (latency: F(1, 30) = 0.67, n.s.; path length: F(1, 30) = 2.88, P = 0.10; CHD rate: F(1, 30) = 7.35, P < 0.05). When the platform was moved in the test at 12 months of age, the difference in path length and CHD rate increased; the restricted rats performed more poorly than the ad lib rats (latency: F(1, 30) = 1.48, n.s.; path length: F(1, 30) = 5.62, P < 0.05; CHD rate: F(1, 30) = 24.84, P < 0.001). The main effects of blocks were significant for these three indices at all
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
329
Fig. 4. Mean percent correct for the spatial discrimination task by glucose-treated restricted group, saline-treated restricted group, and saline-treated ad lib group. The performance of the saline-treated restricted group remained at the chance level, whereas the glucose-treated restricted and ad lib groups learned the task. Vertical bars indicate S.E.M.
3.2. DMTP task (17 months) Ten out of 12 ad lib rats and 3 out of 15 restricted rats achieved the criterion (χ2 (1) = 10.71, P < 0.001). If a subject did not reach the criterion after 40 trials of training, it was given a score of 40. The mean number of trials required to reach the criterion of acquisition was 36.7 cm/sec (S.E.M.: 2.09) for the restricted group and 21.7 cm/sec (S.E.M.: 3.53) for the ad lib group. A t test showed that the mean number of trials to the criterion in the restricted group was larger than that of the ad lib group (t(25) = 3.85, P < 0.001). 3.3. Spatial discrimination task (24–27 months)
Fig. 3. The performance in the Morris-type place task conducted at 2 (prior to the restriction), 3, 7, 9, and 12 months of age: (A) escape latency; (B) swim path length; (C) CHD rate. Because of a defect in the video-recorded images, the swim path length and CHD percentage at the first block of the 7-month retraining are not shown. Vertical bars indicate S.E.M.
retraining sessions except for the CHD rate at the 9 months session. The interaction was significant only for path length at the 7 months session (F(2, 60) = 6.54, P < 0.01). Tests for simple effect showed a significant group effect at the second and fourth blocks (F(1, 90) = 27.51 and 7.86, respectively, P < 0.01). The mean swim speed throughout the retraining sessions of the place task (3, 7, 9, and 12 months sessions) was 24.8 cm/sec (S.E.M.: 1.26) for the restricted group and 20.6 cm/sec (S.E.M.: 1.13) for the ad lib group. A t test confirmed that the restricted group swam significantly faster than the ad lib group (t(30) = 2.45, P < 0.05).
The mean percent of correct responses in four six-trial blocks in the restricted-glucose group (Restricted-Glu), the restricted-saline group (Restricted-Sal), and the ad lib-saline group (Ad lib-Sal), respectively, are presented in Fig. 4. The performance of the restricted-saline group remained at the chance level, whereas the mean percentage of correct responses increased in the restricted-glucose and ad lib-saline groups. An ANOVA revealed a significant group effect (F(2, 15) = 5.05, P < 0.05) and trial-block effect (F(3, 45) = 5.36, P < 0.01). Student–Newman–Keuls multiple range test on the groups revealed a significant difference between the restricted-saline groups and the other two groups (P < 0.05). The interaction between the group and the trial blocks was not significant (F(6, 45) = 1.99, P < 0.10). 3.4. Blood glucose level and body weight The mean blood glucose level and the mean body weight up to 23 months of age are presented in Fig. 5. After the
330
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
Fig. 5. Mean blood glucose level (GLU) and mean body weight (BW) of restricted and ad lib rats. Figures under the age (month) show the number of subjects alive at each age. Vertical bars indicate S.E.M. Except for Month 2.5, there were significant differences between the ad lib and restricted groups every month in both the body weight and the blood glucose level.
beginning of dietary restriction, the blood glucose levels in the restricted rats were consistently lower than those in the ad lib rats, and were maintained at a stable low level. An ANOVA was conducted for the glucose data taken in a period from 2.5 to 11 months of age, when all subjects were alive. The analysis revealed a significant group effect (F(1, 30) = 274.87, P < 0.001), age effect (F(5, 150) = 23.94, P < 0.001), and interaction (F(5, 150) = 14.73, P < 0.001). The simple effect of group was significant at all ages (P < 0.001) except at 2.5 months of age. There was no difference in body weight between the groups at 2.5 months of age, but after the beginning of dietary restriction the mean body weight of the restricted rats remained stable at 280 g. The body weight of the ad lib rats increased until about 10 months of age, and then declined at 23 months. An ANOVA for the body weight using the data of the 2.5, 3, 5, 7, 9, and 11 months of age revealed a significant group effect (F(1, 30) = 401.91, P < 0.001), age effect (F(5, 150) = 548.70, P < 0.001), and interaction (F(5, 150) = 518.85, P < 0.001). There was a significant simple effect of age for the ad lib rats (F(5, 150) = 39.40, P < 0.001) and a group effect after 3 months (P < 0.001).
4. Discussion The effect of dietary restriction on longevity is well established. In order to examine the effect of dietary restriction and aging on the acquisition and performance of various tasks in addition to longevity, we gave our subjects spatial tasks that were independent of food motivation. The life span of the restricted rats was longer than that of the ad lib rats, but the restricted rats showed overall impairments in the tasks. In the Morris-type place task, the path length became longer and the CHD rate became lower in
the restricted rats at the age of 7 months and thereafter. In addition, the restricted rats performed poorly on the place task when the platform was moved, and also on the DMTP task and the spatial discrimination task. Altogether, these results suggest that dietary restriction has a negative effect on spatial cognition, despite a beneficial effect on longevity. It has been reported that dietary restriction either eliminates age-related learning impairments [12,13] or has no effect [2,3,5,19]. The results of the present study are not congruent with any of these findings. The discrepancy between the previous studies and our study may be explained by the differences in the tasks and the motivational level. In the studies using food-motivated tasks, the ad lib rats had to be placed under dietary restriction before the beginning of the training [2,5,12]. Because of this procedure, it is unclear if the reward value was equivalent for the restricted group and the ad lib group, making the findings from these studies unreliable. The studies that used aversive tasks were also different from ours in procedure. In a study by Gyger et al., a Morris-type pool had four panels on the rim. These panels could have served as a kind of proximal cue, making the type of the task unclear [13]. The strain of the rats used by Markowska was different from ours [19]. The extent of the dietary restriction was also different. The extent of our dietary restriction (to maintain the body weight of the restricted rats at 280 g; almost 35% of the maximum body weight of ad lib rats) was more stringent than the most of the previous researches. The body weight of the restricted rats in a study by Markowska was maintained at 60% of the ad lib counterparts [19]. These procedural differences may have caused the inconsistency in results. It has been reported that the mean life span of Wistar rats, the same strain used in the present study, is 785 days [8]. In the present study, 3 of 16 ad lib rats were alive when they were 785 days of age. Although the life span of our ad
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
lib rats seems shorter than that in the above report [8], the average life span of our restricted rats seems much longer than 785 days, because 13 out of our 16 restricted rats were alive at 785 days. Thus, our results on the beneficial effect of dietary restriction on longevity agree with the findings of other studies [21,36]. In the retraining of the place task, no difference was seen between the restricted and the ad lib rats in the escape latency except for the 7 months retraining. However, the analysis of the swim path length revealed that the restricted rats swam longer than the ad lib rats at the 7 months session and thereafter. Moreover, the calculated swim speed showed that the restricted rats swam faster in all sessions, though statistical significance was shown only at the 3 and 9 months sessions. The faster swim by the restricted rats may have compensated for their cognitive deficiency: as a result, their escape latencies nearly matched with those of the ad lib rats in most of the sessions. Considering this, the CHD rate may be a better index for assessing cognitive ability than the escape latency in the case of the present study. The mean CHD rate in the restricted group was consistently lower than that in the ad lib group at the 7-month test and thereafter. In addition to that, the restricted group swam longer and the mean CHD rate was lower than that in the ad lib group on the place task with platform moved at 12 months. Altogether, the spatial learning ability of the restricted rats is poorer than the ability of the ad lib rats in the acquisition of the new platform position as well as in the retention of the platform position. Our dietary restriction schedule was to maintain the body weight at 280 g throughout the experiment period. This schedule was more stringent than in some other studies [3,13,19], though there are some researchers who imposed similar [5] or more stringent restriction [34]. It is possible that dietary restriction may have resulted in the low body temperature in the restricted rats; we did not measure the body temperature. The low body temperature may have aggravated when the restricted rats were immersed in water. However, the water temperature was maintained at 23±1 ◦ C while the room temperature was 24 ◦ C. Moreover, the rats were dried with a towel as soon as they were out of the pool. In spite of these efforts to minimize the dissipation of body heat, we cannot exclude the possibility that the conceivable thermogenic differences resulting from dietary restriction could contribute to the impaired performance of the restricted rats. The Morris-type place task is a spatial task that requires reference memory. In this task, the platform remains in the same position throughout the trials. The restricted rats performed poorly in this task at the age of 7 months and thereafter. The spatial discrimination task also depends on reference memory since the rats have to discriminate between the true platform, which remained stationary, and the false one, which moved from one trial to another. In this task, the restricted rats without glucose injection showed very poor performance. The DMTP task is a spatial task that
331
requires working memory: the rats have to retain the memory of the platform position during the delay period. The restricted rats performed very poorly in this task. Theses results indicate that the restriction-induced deficit generalizes to more than one type of spatial memory. Altogether, our experiments suggest that the dietary restriction interferes with the spatial cognitive ability in aged rats. The blood glucose level in the restricted rats was stable and consistently lower than that in the ad lib rats. Since the blood glucose level is thought to reflect the glucose level in the brain [14], the impaired performance of the restricted rats may have been caused by the decreased amount of glucose taken up by the brain. This interpretation receives a strong support from our glucose injection data. Intraperitoneal injection at the age of 24–27 months improved the performance of the restricted group in the spatial discrimination task to the level of the ad lib group. Although many studies reveal positive effect of glucose injection [17,23,37,38], it is still unknown how administered glucose functions. We need further research on this matter.
Acknowledgments The authors are grateful to the editor and the reviewers for their helpful comments. This research was supported by the Scientific Frontier Project of Doshisha University, which was subsidized by the Ministry of Education, Culture, Sports, Science and Technology.
References [1] Barnes CA. Memory changes during normal aging: neurobiological correlates. In: Martinez J, Kesner R, editors. Neurobiology of learning and memory. San Diego, CA: Academic Press; 1998. p. 247–87. [2] Beatty WW, Clouse BA, Bierley RA. Effects of long-term restricted feeding on radial maze performance by aged rats. Neurobiol Aging 1987;8:325–7. [3] Bellush LL, Wright AM, Walker JP, Kopchick J, Colvin RA. Caloric restriction and spatial learning in old mice. Physiol Behav 1996;60:541–7. [4] Berg BN, Simms HS. Nutrition and longevity in the rat. III. Food restriction beyond 800 days. J Nutr 1961;74:23–32. [5] Bond NW, Everitt AV, Walton J. Effects of dietary restriction on radial-arm maze performance and flavor memory in aged rats. Neurobiol Aging 1989;10:27–30. [6] Caprioli A, Ghirardi O, Giuliani A, Ramacci MT, Angelucci L. Spatial learning and memory in the radial maze: a longitudinal study in rats from 4 to 25 months of age. Neurobiol Aging 1991;12:605–7. [7] Evans GW, Brennan PL, Skorpanich MA, Held D. Cognitive mapping and elderly adults: verbal and location memory for urban landmarks. J Gerontol 1984;39:452–7. [8] Everitt AV, Seedman NJ, Jones F. The effects of g and continuous food restriction, begun at ages 70 and 400 days, on collagen aging, proteinuria, incidence of pathology and longevity in the male rat. Mech Ageing Dev 1980;12:161–72. [9] Frick KM, Price DL, Koliatsos VE, Markowska AL. The effects of nerve growth factor on spatial recent memory in aged rats persist after discontinuation of treatment. J Neurosci 1997;17:2543–50.
332
S. Yanai et al. / Neurobiology of Aging 25 (2004) 325–332
[10] Gage FH, Dunnet SB, Björklund A. Age-related impairments in spatial memory are independent of those in sensorimotor skills. Neurobiol Aging 1989;10:347–52. [11] Gallagher M, Burwell RD. Relationship of age-related decline across several behavioral domains. Neurobiol Aging 1989;10:691–708. [12] Goodrick CL. Effects of lifelong restricted feeding on complex maze performance in rats. Age 1984;7:1–3. [13] Gyger M, Kolly D, Guigoz Y. Aging, modulation of food intake and spatial memory: a longitudinal study. Arch Gerontol Geriatr Suppl 1992;3:185–96. [14] Harada M, Sawa T, Okuda C, Matsuda T, Tanaka Y. Effects of glucose load on brain extracellular lactate concentration in conscious rats using a microdialysis technique. Horm Metab Res 1993;25:560–3. [15] Jucker M, Oettinger R, Bättig K. Age-related changes in working memory and reference memory performance and locomotor activity in the Wistar rat. Behav Neural Biol 1988;50:24–36. [16] Kirk RE. Experimental design, 2nd ed. Belmont, CA: Wadsworth; 1982. [17] Kopf SR, Baratti C. Memory modulation by post-training glucose or insulin remains evident at long retention intervals. Neurobiol Learn Memory 1996;65:189–91. [18] Maki PM, Zonderman AB, Weingartner H. Age differences in implicit memory: fragmented object identification and category exemplar generation. Psychol Aging 1999;14:284–94. [19] Markowska AL. Life-long diet restriction failed to retard cognitive aging in Fischer-344 rats. Neurobiol Aging 1999;30:177–89. [20] Markowska AL, Price D, Koliatsos VE. Selective effects of nerve growth factor on spatial recent memory assessed by a delayed nonmatching-to-position task in the water maze. J Neurosci 1996;16:3541–8. [21] McCay CM, Crowell F, Maynard LA. The effect of retarded growth upon the length of life span and upon the ultimate body size. J Nutr 1935;18:63–79. [22] Means LW, Kennard KJP. Working memory and the aged rat: deficient two-choice win-stay water-escape acquisition and retention. Physiol Behav 1991;49:301–7. [23] Messier C, Destrade C. Improvement of memory for an operant response by post-training glucose in mice. Behav Brain Res 1988;31:185–91. [24] Mizumori SJY, Kalyani A. Age and experience-dependent representational reorganization during spatial learning. Neurobiol Aging 1997;18:651–9.
[25] Morris RGM. Spatial localization does not require the presence of local cues. Learn Motiv 1981;12:239–60. [26] Okaichi H. Effects of dorsal–striatum lesions and fimbria–fornix lesions on the problem-solving strategies of rats in a shallow water maze. Cognit Affect Behav Neurosci 2001;1:229–38. [27] Okaichi Y, Okaichi H. Effects of glucose on scopolamine-induced learning deficits in rats performing the Morris water maze task. Neurobiol Learn Memory 2000;74:65–79. [28] Olton DS, Becker JT, Handelmann GE. Hippocampus, space, and memory. Behav Brain Sci 1979;2:313–65. [29] Rapp PR, Rosenberg RA, Gallagher M. An evaluation of spatial information processing in aged rats. Behav Neurosci 1987;101: 3–12. [30] Sharps MJ, Gollin ES. Memory for object locations in young and elderly adults. J Gerontl 1987;42:336–41. [31] Sohal RS, Agarwal S, Candas M, Forster MJ, Lal H. Effect of age and caloric restriction on DNA oxidative damage in different tissues of C57BL/6 mice. Mech Ageing Dev 1994;76: 215–24. [32] Sohal RS, Ku HH, Agarwal S, Forster MJ, Lal H. Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech Ageing Dev 1994;74:121–33. [33] van der Staay FJ. Shift in the performance of 24-month-old Wistar rats in the Morris water escape task: a comparison across 36 experiments. Behav Brain Res 1997;87:213–22. [34] Wada H, Hosokawa T, Saito K. Single toluene exposure and changes of response latency in shock avoidance performance. Neurotoxicol Teratol 1989;11:265–72. [35] Walford RL, Liu RK, Gerbase-Dekima M, Mathies M, Smith GS. Long-term dietary restriction and immune function in mice: response to sheep red blood cells and to mitogenic agent. Mech Ageing Dev 1973;2:447–54. [36] Weindruch R, Walford RL. Dietary restriction in mice beginning at 1 year of age: effects on life-span and spontaneous cancer incidence. Science 1982;215:1415–8. [37] Winocur G. Glucose-enhanced performance by aged rats on a test of conditional discrimination learning. Psycobiology 1995;23: 270–6. [38] Winocur G, Gagnon S. Glucose treatment attenuate spatial learning and memory deficits of aged rats on tests of hippocampal function. Neurobiol Aging 1998;19:233–41.