Physiology&Behavior,Vol. 51, pp. 1001-1007, 1992
0031-9384/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.
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Spatial Learning Ability of Rats Undernourished During Early Postnatal Life K. S. B E D I
Department of Anatomy, University of Queensland, St. Lucia, Brisbane, Queensland, Australia, 4072 Received 16 A u g u s t 1991 BEDI, K. S. Spatial learning ability of rats undernourished during early postnatal life. PHYSIOL BEHAV 51(5) 1001-1007, 1992.--Experiments to determine whether undernutrition during early life results in deficits in spatial learning behavior has produced conflicting results. It was hypothesized that this may be due to the differing degrees of undernutrition used in the various studies, and/or to the timing of the testing procedures with respect to the period of food deprivation. These possibilities were tested by undernourishing rats between birth and 30 days of age to two different levels (i.e., level-I and level-2). The degree of undernutrition was greater at level-2 than level-l. Behavioral testing of these rats and well-fed controls was carried out in the Morris water maze when they were between either 35 and 65 or 170 and 200 days of age. Statistical analyses of the escape latency data from these experiments revealed that rats tested almost immediately after the period of undernutrition have alterations in their spatial learning behavior compared with controls. However, even a short period of nutritional rehabilitation removed any differences between control and previously undernourished groups, irrespective of the level of undernutrition. Malnutrition
Behavior
Memory
STUDIES in which the rat hippocampal formation or its connections have been lesioned have demonstrated that it is an important brain center influencing, among other things, the spatial learning capacity of the animals (l l, 19,20,25). Such lesioned animals show deficits in spatial learning capacity as demonstrated by a number of behavioral tests. Spatial learning behavior of rats is often tested using 8-, 12-, or 16-radial arm mazes [e.g. (14,15)] and the Morris water maze test (18,19). Undernutrition of rats during early postnatal life is known to cause changes in the developing brain. These alterations are not characterized by qualitatively visible lesions, but instead consist of quantitative deficits and distortions (10) of brain structure (3,4,6). A brain region particularly affected by undernutrition during early life is the hippocampal formation [e.g. (1,2,5,9)]. This observation has led to a small number of behavioral studies (5,7,12-16) designed to test the spatial learning ability of rats undernourished during early life. The results of these studies are somewhat conflicting. For example, Jordan et al. (14) examined 90-day-old rats previously undernourished during the gestation and suckling periods. The performance of these rats was significantly different from controls in a radial maze task. However, Hall (13), who used similar behavioral methods in three separate experiments, could not confirm the results obtained by Jordan et at. (14). The use of radial arm mazes to test the spatial learning ability of undernourished rats is problematical. A food reward (reinforcement) is required in order to induce the rats to perform the test. In order to accomplish this, the rats have to be hungry at the time of testing. This is normally achieved by placing the rats on a diet, slightly reduced in amount, in order to decrease the body weight of the rats just prior to testing. This is obviously a complicating factor in experiments where malnutrition or un-
1001
dernutrition is the factor under investigation in the first place. The problem is further exacerbated because there is now considerable evidence (22,23) that rats previously undernourished during early life have differential degrees of motivation to obtain rewards (particularly food or water reinforcements) compared with well-fed rats. Jordan et ai. (14) did not attempt to account for this factor. Hall (13) attempted to equate motivational levels of his nutritional groups by reducing the body weights of the controls and experimental rats to different degrees just prior to the testing procedures. However, there is no evidence presented that this maneuver was successful in equating levels of motivation. The use of the Morris water maze (18) for testing spatial learning ability in undernourished rats offers the advantage that an appetitive reward is not required. This circumvents some of the problems mentioned above. Previously published studies that have used the Morris water maze test on undernourished rats are those by Jordan and Taghavi (16), Goodlett et al. (12), Castro and Rudy (8), and Campbell and Bedi (7). Of these, the study by Goodlett et at. (12) only examined adult rats that had been undernourished from conception and throughout their lives. Castro and Rudy (8) only tested young rats, almost immediately following the period of undernutrition. The details of these studies are discussed further in this paper. The previous study carried out in my laboratory (7) examined adult rats that had been undernourished during early postnatal life but then allowed a lengthy period of nutritional rehabilitation before behavioral assessment. The length of the period of undernutrition was either 30 or 60 days and commenced at birth. No statistically significant deficits could be demonstrated in the spatial learning ability of control and experimental animals. However, the coefficients of variation between individual animals
1002
BEDI
within a given group were sufficiently large to make it uncertain whether a type It error (24) had not occurred. In the present study I have assessed whether the level of undernutrition imposed during early life could be a factor in influencing the spatial learning capacity of the animals. This paper describes the results of tests carried out on animals soon after the end of the period of undernutrition, as well as on rats given a lengthy period of nutritional rehabilitation before behavioral testing. METHOD
Animals The rats used in this study were of an outbred hooded LongEvans strain purchased from the Central Breeding Unit of Monash University, Victoria. Virgin females were housed overnight with males and the presence of a vaginal plug the next day was used to determine whether mating had occurred. Mated females were housed in separate cages. These dams were divided, at random, into three groups on the day 16 of pregnancy. The first group was designated as the control group; the second and third as the level-1 and level-2 undernourished groups, respectively. At birth all litters were standardized, by culling, to contain eight pups with at least two, but not more than four, females in each litter. There was no crossfostering of any pups between litters. The control animals and their offspring were allowed to feed ad lib throughout the experiment. The amount of food given to the level-l and level-2 dams was restricted to that shown in Table I from the day 16 of pregnancy and throughout the suckling period. All male pups were weaned on postnatal day 19 by separating them from their mothers and housing them three to a cage. Female pups were euthanized at this stage. The amount of food given to level-I and level-2 pups was restricted to the values shown in Table l until they had reached 30 days of age. The daily food intake of the level-l and level-2 rats represented about 60% and 40% of that eaten by the well-fed control animals at the same stage. Water was available to all rats ad lib throughout the experiment. Nutritional rehabilitation of the undernourished pups was commenced when they had reached 30 days of age by placing them on an ad lib diet.
Experiment 1 This was carried out on rats between 35 and 65 days of age, i.e., soon after the end of the period of undernutrition. There were 15 control and 15 level- 1 undernourished rats, each group being derived from five separate litters. There were 11 level-2 undernourished rats derived from six litters. All rats were between 35 and 65 days of age during the testing period. Because it was only possible to test about 15 rats on any given day, the available rats were subdivided into three subgroups. The first subgroup contained three control, five level- 1, and four level-2 rats. The second subgroup contained six control, four level-1, and five level-2 rats. The third subgroup contained six control, six level- 1, and two level-2 rats. The rats within a given nutritional group in these subgroups were, as far as possible, derived from separate litters. One of the subgroups was tested during each of the first 3 weeks of the test period. The order in which the individual rats were tested during a given week was randomized between nutritional groups. The testing apparatus consisted of a circular tank of diameter 1.53 m and height 0.31 m filled with water at a temperature of 25 +_ 1°C to a depth of 0. ! 8 m. The water was made opaque by the addition of about two liters of milk. The tank was located
TABLE 1 DAILY A M O U N T S (g) OF FOOD GIVEN l'O RATS D U R I N G EARLY DEVEI_.OPMENT I Indernourished Rats
Gestation G 16-birth Postnatal PND0-PND6 PND7-PND t 3 PNDI4-PNDI8 Post-weaning PND 19-PND21 PND22-PND25 PND26-PND30
Level- I
I cvel-2
12
8
17 22 27
13 18 23
3 3.5 4
2 2.5 3
G, Gestation. PND, Postnatal day. Note that during the gestation and lactation periods, it was the dam that received the food intake indicated, while after weaning it was the pups that received the food indicated(see Method Section for detailed description). in the center of a test room, the floor area measured 2.4 x 3 m, the ceiling being 2.4 m high. The walls of this room had no windows; the door leading into the adjoining room was fitted with a black curtain. A closed circuit video camera, equipped with a wide angle lens, was fitted in the center of the ceiling of the test room, directly above the pool. Other equipment placed within the test room provided distinct visual cues. The closed circuit video camera relayed a picture of the pool to a video tape recorder and monitor in the adjoining room. The image of the pool on the monitor was marked into four equal quadrants labeled N, S, E, and W. Four equally spaced start points were marked around the circumference of the pool, one for each quadrant. A 14 cm diameter platform was constructed with perspex and covered on the outside with a white colored cotton material. The height of this platform was 0.17 m so that when it was placed in the pool of water it became completely submerged to a depth of about 1 cm below the surface. The white material helped to make the platform invisible in the milky-colored water in the pool.
Spatial Learning Test--Escape Acquisition There was no pretraining period for any of the rats. The main escape acquisition test consisted of eight trials per rat per day over 3 consecutive days. Each trial consisted of placing the rat in the water at one of four starting points, such that it faced away from the center of the pool. The starting point for any given trial was chosen at random. The rats were required to swim until they found, and climbed onto the submerged platform; the time taken (escape latency) to do this for each trial being recorded. The platform was located in the same place (the approximate center of one of the quadrants) for all trials for any given rat. However, this location was randomized between rats. Once the rat had climbed onto the platform in any given trial it was left there for a period of 60 seconds before being removed to a holding cage for a further 1 to 2 minutes. After this, the rat was subjected to the next trial. All trials were recorded on the video tape recorder in the adjoining room from where the trials were monitored by the experimenter.
SPATIAL LEARNING IN UNDERNOURISHED RATS
If a rat failed to find the platform within 6 minutes of the start of any given trial the rat was removed from the pool and placed in the holding cage for a period of between 2 and 5 minutes before the next trial. Out of the 1066 separate trials in this experiment failure to reach the platform in the allotted time only occurred a total of I 1 times and involved two level-I and three level-2 undernourished rats. All 11 failures occurred during the first test day.
1003 using a repeated measures ANOVA for unequal sample sizes, and examined differences between Groups and Test Days (with all three weeks combined) as well as the interaction between them. All statistics were carried out on an IBM-compatable personal computer using a SAS statistical software package (SAS Institute Inc., Cary, NC 27512-8000) on license to the University of Queensland.
Memory Persistence Test Following the initial 3-day testing period, each rat was given one additional trial on each of days 4 and 11 of its test period. For any given rat, the platform remained in the same quadrant as that used in the escape acquisition task.
Experiment 2 This was carried out using rats between 170 and 200 days of age, i.e., after a lengthy period of nutritional rehabilitation. The testing procedures were similar to those used in Experiment 1 except as detailed below. There were ten control, nine level-1, and seven level-2 rats, each group being derived from four separate litters. As these older rats were somewhat larger and heavier than those used in Experiment 1, the spatial learning task was carried out in a larger pool with a diameter of 1.82 m and height of 0.61 m. The maximum time allowed for a rat to find the platform during any given trial was 5 minutes. Out of the 624 trials in experiment 2, failure to reach the platform in the allotted time only occurred a total of 14 times and involved four control, four level-1, and two level-2 rats. Thirteen of these failures occurred on the first test day; the remaining one occurred on the second test day and involved a control rat. In Experiment 2 the memory persistence test was carried out on day 18 as well as on days 4 and 11.
Statistics Body weight data was analyzed by two-way ANOVA procedures. Escape latency data was not normally distributed due to the fact that on occasions some rats took considerably longer to locate the platform than others, causing a degree of positive skewness. A log~otransformation (24) was, therefore, carried out on this data before further univariate analysis. (The number of replications was not considered sufficient to make multivariate analysis appropriate.) As the rats in Experiment 1 were only between 35 and 65 days of age, rats tested at the beginning of the test period could give markedly different results to those rats tested at the end of the test period. Even a few extra days of nutritional rehabilitation at this relatively young age could affect the performance of the animals; l or 2 weeks represents a large portion of the lifespan of 35-65-day-old rat. Therefore, for Experiment l the univariate analysis involved repeated measures ANOVA for unequal sample sizes with groups, weeks (i.e., weeks l, 2, or 3 during test period), days (days l, 2, or 3 during test week), and trials as the variables. This analysis used Huyhn-Feldt's adjusted probabilities necessary because of the repeated measures design of the experiment (17). In Experiment 2 all the rats were between 170 and 200 days of age; the undernourished groups having been nutritionally rehabilitated for a lengthy period of time commencing at 30 days of age. Testing such rats at the beginning or end of the test period would not be expected to greatly influence the results; 1 or 2 weeks in the life of a 200-day-old rat is a relatively short period of time. Therefore, the data in Experiment 2 was analyzed
RESULTS
Body Weights The mean body weights for the three groups of rats in both Experiments ! and 2 are presented in Table 2, together with the results of two-way ANOVA of the data. There were significant deficits in mean body weight between the three groups of rats at all ages examined. As expected, the well-fed control rats were substantially heavier than the level-t and level-2 undernourished rats. However, it was also noted that the level-1 rats were, in turn, significantly heavier than level-2 rats. Furthermore, nutritional rehabilitation after 30 days of age did not seem to restore the deficits in body weight for either level-1 or level-2 undernourished rats (Table 2).
Escape Latency Figures 1 and 2 show the mean escape latency for each trial and nutritional group for the escape acquisition task plotted on a semilog scale for Experiments 1 and 2, respectively. These show that all three groups of rats were able to learn to locate the position of the platform after a few trials. Table 3 shows the logm latency data tabulated to show group means by day and week for Experiment 1, and group means by day (with all three weeks combined) for Experiment 2. This data was subjected to an overall repeated measures ANOVA for unequal sample sizes. The results of these analyses are shown in Table 4. Examination of the data from Experiment 1 shows differences between nutritional groups during week 1, but not during weeks 2 and 3. During week 1 the undernourished rats appear to take longer to find the platform than control animals; in all cases the control rats mean logtime taken is less than that
TABLE 2 MEAN _+ SEM BODY WEIGHTS(g) OF RATS USED IN THE BEHAVIORAL TESTINGNEAR THE BEGINNING AND END OF THE TEST PERIOD
Experiment 1 Number in group Age (days) 30 60 Experiment 2 Number in group Age (days) 165 200
Control Rats
Level-1Rats
Level-2Rats
15
15
11
90.6 _+ 1.5 243.4 _+4.1
34.5 _+0.4 189.7 _+5.6
23.5 _+ 1.0 160.7 _+ 4.2
II
9
7
378.9 +_9.2 395.8 _+8.1
317.7_+6.1 336.2 _+6.9
302.9 _+ 12.1 307.0+ 13.9
Results of two-way ANOVA of above data: Experiment 1: age, p < 0.0001; nutrition, p < 0.0001; age X nutrition, p < 0.01. Experiment 2: age, p < 0.05; nutrition, p < 0.001; age X nutrition, NS.
1004
BI-~DI 10 .... ,D--- Control
........t ....... Level 1 o----
Level 2
J
0.1
I
5
I
!
10
!
15
20
25
Trial number
FIG. 1. Plots to show the mean escape latencies against a given trial number for the three groups of rats in Experiment 1. There were eight trials per day for 3 days for each rat. The effects of test week (see Method section) have been ignored for this plot. Each point represents the mean value for all the rats in a given group for a given trial number. Standard error bars are also shown. Experiment 1 was carried out on rats between 35-65 days of age. for the level- 1 and level-2 rats. This is not so, however, for weeks 2 and 3 (Table 3). These differences are reflected in the ANOVA test by a significant main effect of groups as well as a significant week X groups interaction (Table 4). In other words, the differences between the three nutritional group means vary significantly depending on whether or not the experiment was done in week 1, 2, or 3 of the test period. The mean escape latencies for all groups decreased over the three test days during any given week. These observations were reflected by a significant main effect of days and days × week interaction in the ANOVA test (Table 4). The days × group and days X week X group interactions were not statistically significant. In other words, the differences between the means for the 3 days (for all nutritional groups) varied to some extent depending on the week of the experiment. There was also a significant main effect of trials and a days x trials interaction (Table 4). All the remaining first, second, and third order interactions were not significant (Table 4). These results indicate that the intertrial variation itself varies depending on the day of the experiment, and that this intertrial variation may be significant to a greater or lesser degree, depending on the day of the experiment. This was irrespective of the nutritional group or the week of the experiment. In Experiment 2 there were no significant differences between groups, although there was significant effects of trials and days
(Table 4). The days X groups and trial X groups interactions were also not significant (Table 4).
Memory Persistence Test Table 5 shows the mean escape latencies for the groups of rats on the single trials carried out on days 4 and 11 for Experiment 1 and days 4, I 1, and 18 for Experiment 2. In Experiment 1, two-way ANOVA with the day of testing and nutrition as the two factors revealed a significant main effect of days but not nutrition. The day X nutrition interaction was also not statistically significant. These results are reflected in the observation that all groups of rats appeared to take significantly longer to locate the platform on day 11 than they did on day 4 (Table 5). The results for the memory persistence test in Experiment 2 were broadly similar to those obtained in experiment 1, and are also summarized in Table 5. G E N E R A L DISCUSSION In Experiment 1, the rats tested in weeks 2 and 3 had slightly longer periods of nutritional rehabilitation than those tested in week 1, that were in effect tested between 6 and 9 days following the end of the period of undernutrition. At the relatively young age that these rats were tested, even such small differences in age and degree of nutritional rehabilitation can be conceived to
SPATIAL LEARNING IN UNDERNOURISHED RATS
1005
10
!i
.... • ....
Control
#-
Level 1
o---
Level 2
1
0.1
I
0
5
I
10
I
|
15
20
25
Trial number
FIG. 2. Plots to show the mean escape latencies against a given trial number for the three groups of rats in Experiment 2. There were eight trials per day for 3 days for each rat. The effectsof test week (see Method Section) have been ignored for this plot. Each point represents the mean value for all the rats in a given group for a given trial number. Standard error bars are also shown. Experiment 2 was carried out on rats between 170-200 days of age. markedly effect their performance in behavioral tasks. The results of this experiment showed significant intergroup differences and a significant week × group interaction. In all cases, the mean log,o latency for the controls was less than that for the level-1 and level-2 rats during week 1. However, by weeks 2 and 3 the differences were not so obvious and were not significant, although in all cases the level-2 rats took marginally longer to find the platform. These results indicate that even a short period of nutritional rehabilitation can remove any differences in spatial learning capacity between nutritional groups. This finding was further confirmed in Experiment 2, where the rats were given a lengthy period of nutritional rehabilitation before being tested in the Morris water maze. The results of this experiment showed that there were no statistically significant long term effects on the performance of these rats, compared with age-matched controls, in the spatial learning task used. This finding is in agreement with our previous research (7). Our results indicate that all groups of rats were capable of learning to find the hidden platform over the 3-day test period of the experiment, usually by the end of the first day. Having learned to find the platform, little or no further improvement would be expected on days 2 and 3. This, in fact, seems to be the case for most groups of rats in both Experiments 1 and 2. The memory persistence tests revealed that the rats took slightly but significantly longer to locate the platform on day 11
than on day 4 in Experiment 1 and on days 11 and 18 than on day 4 in Experiment 2. Whether this indicates a marginal loss of memory between these days is not of great interest. What is important is that both control and undernourished rats appeared to behave in the same way in this respect. It should be mentioned that proximal-cue navigation using a visible platform was not tested in the present experiments. In previous work using rats given a lengthy period of nutritional rehabilitation following undernutrition during early postnatal life we (7) could not find any differences in proximal-cue navigation between control and experimental rats. The experiments in this previous work were of a similar design to that in Experiment 2 in the present study. It was, therefore, felt to be unnecessary to repeat the proximal-cue navigation test in Experiment 2. It would, however, have been useful to test proximal-cue navigation in rats soon after the period of undernutrition (i.e., those in Experiment 1). Such a test may have served as a kind of pretraining period insofar as the rats could have learned that escape was possible by finding a platform, making interpretation of the results more difficult. Unfortunately, the number of rats available in the present study precluded the possibility of carrying out the proximal-cue navigation test on a separate subgroup of rats than those used in the main escape acquisition test. It was considered best to concentrate all the available animals into the crucial distal-cue navigation test, thereby increasing the likeli-
1006
BEDI TABLE 3 GROUP MEANS OF Iog~o LATENCY BY DAY AND WEEK Day 1 Experiment 1 Week 1 Control [24] Level- 1 [40] Level-2 [32] Week 2 Control [48] Level- 1 [32] Level-2 [40] Week 3 Control [48] Level- 1 [48] Level-2 [ 16] Experiment 2* Control [80] Level- 1 [72] Level-2 [56]
Day 2
Day 3
1.103 1.709 1.680
0.862 1.357 1. I 13
0.720 0.926 1.007
1.162 1.124 1.459
1.033 0.994 1.055
0.832 0.855 0.981
1.315 1.228 1.401
0.930 0.926 1.108
0.849 0.914 1.061
1.598 1.495 1.573
1.151 1.249 1.225
0.963 1.038 1.067
Number of trials are in brackets. * In this experiment the data was analyzed with weeks 1, 2, and 3 combined.
hood of detecting any differences in escape latencies between nutritional groups, had they existed. Spatial learning ability of undernourished rats has been previously investigated using the Morris water maze (7,8,12,16). Goodlett et al.'s (12) experiments were designed to examine adult rats that had been undernourished from conception and throughout their lives. They concluded that chronic malnutrition in rats did not impair spatial localization ability and spatial memory processes. However, the malnourished rats had a somewhat slower rate of learning than controls. The results of Goodlett et al.'s (12) experiment are not directly comparable with our present work. Long term and ongoing malnutrition during the test period, like that used by Goodlett et al. (12), may well result in different behavioral deficits than those induced by a short period of undernutrition during early life. The study by Castro and Rudy (8) is more directly comparable to the present investigations. They examined 22- to 30-day-old rats that had been undernourished between postnatal days 2 and 18. In their Morris maze tests they employed either a visible platform (proximal-cue navigation) protruding just above water level or a hidden platform (distal-cue navigation) the surface of which was just below the level of the opaque water in the pool. They concluded that proximal-cue navigation was not influenced by nutrition. This finding was confirmed by us (7) in previously reported experiments. Castro and Rudy (8) also found that malnutrition only had a mild influence on distal-cue navigation (i.e., hidden platform test) at 30 days of age. However, 22-27day-old rats had significantly worse distal-cue navigation than control animals. In Experiment l with the hidden platform, rats were also tested fairly soon after a period ofundernutrition during early life. As mentioned above, the results indicate that there are initial differences between nutritional groups in the performance in the Morris water maze. These findings, therefore, go to support the observations made by Castro and Rudy (8). However, it can be argued that the performance of rats tested soon after a period of undernutrition could be influenced by their physical weakness rather than by, or in addition to, any
TABLE 4 OVERALL REPEATED MEASURES UNIVARIATE ANOVA ()t:: log~o Source Experiment 1 Group Week Week × group Error Days Days X group Days X week Days X week × group Error (days) Trial Trial X group Trial X week Trial X week x group Error (trials) Days x trial Days × trial X group Days × trial X week Days × trial X week X group Error (days x trials) Experiment 2 Groups Error Days Days X groups Error (days) Trials Trials X groups Error (trials) Days X trials Days X trials x groups Error (days X trials)
d/
MS
t
,
2 ~ 4 32 2 4 4 8 64 7 14 14 28 224 14 28 28 56 448
3.676 0.956 1.701 0.359 15.324 0.223 0.565 0.280 0.212 3.539 0.099 0.077 0.082 0.096 0.699 0.010 0.085 0.072 0.101
10.25 2.67 4.75
0.0004 0.0849 0.0040
72.36 i.05 2.67 1.32
0.0001 0.3821 0.0492 0.2586
37.06 1.03 0.80 0.86
0.0001 0.4206 0.6655 0.6707
6.96 0.99 0.84 0.72
0.0001 0.4749 0.6973 0.9344
2 23 2 4 46 7 14 161 14 28 322
0.129 1.001 14.895 0.242 0.427 2.091 0.131 0.170 0.287 0.120 0.134
0.13
0.8812
34.93 0.57
0.0001 0.6859
12.28 0.77
0.0001 0.6815
2.14 0.89
0.0126 0,6169
changes in the functioning of the central nervous system. The results of the present study supports this possibility. Any deficits in spatial learning capacity disappeared with progressively longer periods of nutritional rehabilitation. In other words, in Experiment 1, group differences were only observed during week 1 of
TABLE 5 MEAN 4-_ SEM ESCAPE LATENCY (SECONDS) OF RATS IN TRIALS Control Experiment 1 Number in group Day4 Day 11 Experiment 2 Number in group Day4 Day ll Day 18
Level-1
Level-2
15 15 11 6.43_+ 1 . 1 4 8.69_+ 1 . 1 2 11.34_+2.34 22.3 -+ 7.4 23.1 -+ 10.3 21.4 _+7.2 10 13.4 _+ 3.8 19.7 _+ 7.7 30.4 _+ 10.3
9 14.1 -+ 2.3 26.7 _+11.4 16.9 _+ 5.0
7 14.0 _+4.5 23.0 _+7.1 21.7 _+4.9
Results of two-way ANOVA of above data: Experiment 1: day, p < 0.05; nutrition, NS; day × nutrition, NS. Experiment 2: day, NS; nutrition, NS; day x nutrition, NS.
SPATIAL L E A R N I N G IN U N D E R N O U R I S H E D RATS
the test and not during weeks 2 and 3. The results of Experiment 2, which tested rats following a lengthy period of nutritional rehabilitation, showed no intergroup differences. Taken together, these observations supports the hypothesis that the group differences observed in Experiment 1, and those described by Castro and Rudy (8) were transient and not long-term effects of the period of undernutrition. The only studies to have examined previously undernourished rats following a lengthy period of nutritionally rehabilitation with the Morris water maze test were those by Jordan and Taghavi (16) and that carried out in my laboratory (7). In our previous work (7) that employed both a visible and a hidden platform test, we could not demonstrate any statistically significant effect of undernutrition during early life on spatial memory performance. The study by Jordan and Taghavi (16) reported in a short abstract (and, therefore, necessarily devoid of detailed statistical information) found that the persistence of spatial memory of previously undernourished rats was significantly poorer than
1007 well-fed controls. There is no immediately obvious explanation for the discrepancy of this finding with our own present and previous work (7). In conclusion, the present experiments provide evidence that rats tested almost immediately after a period of undernutrition during early life have deficits in their spatial learning behavior compared with well-fed controls. However, there was strong evidence to indicate that nutritional rehabilitation, even for a period as short as 2 or 3 weeks, was capable of removing such deficits. In other words, undernutrition during early postnatal life did not permanently affect the spatial learning behavior of rats, as tested with the Morris water maze test. ACKNOWLEDGEMENTS 1 would like to thank Mr. D. Murray for providing research assistance during this work and Dr. Joan Hendrikz for providing advice and help with the statistical analysis. This research was funded by a grant from the NH and MRC of Australia.
REFERENCES 1. Ahmed, M. G. E.; Bedi, K. S.; Warren, M. A.; Kamel, M. M. The effects of a lengthy period of undernutrition from birth and subsequent nutritional rehabilitation on the synapse-to-granule cell neuron ratio in the rat dentate gyms. J. Comp. Neurol. 263:146-158; 1987. 2. Andrade, J. P.; Cadete-Leite, A.; Madeira, M. D.; Paula-Barbosa, M. M. Long-term low-protein diet reduces the number of hippocampal mossy fibre synapses. Exp. Neurol. 112:119-124; 1991. 3. Bedi, K. S. Effects ofundernutrition on brain morphology: A critical review of methods and results. In: Jones, D. G., ed. Current topics in research on synapses, vol. 2. New York: Alan Liss; 1984:93-163. 4. Bedi, K. S. Lasting neuroanatomical changes following undernutrition during early life. In: Dobbing, J., ed. Early nutrition and later achievement. London: Academic Press; 1-36; 1987. 5. Bedi, K. S. Effects ofundemutrition during early life on granule cell numbers in the rat dentate gyms. J. Comp. Neurol. 311:425-433; 1991. 6. Bedi, K. S.; Warren, M. A. Effects of nutrition on cortical development. In: Peters, A.; Jones, E. G., eds. Cerebral cortex: Development and maturation of cerebral cortex, vol. 7. New York: Plenum; 1988:441-478. 7. Campbell, L. F.; Bedi, K. S. The effects of undernutrition during early life on spatial learning. Physiol. Behav. 45:883-890; 1989. 8. Castro, C. A.; Rudy, J. W. Early life malnutrition selectively retards the development of distal- but not proximal-cue navigation. Dev. Psychobiol. 20:521-537; 1987. 9. Cintra, L; Diaz-Cintra, S.; Galvan, A.; Kemper, T.; Morgane, P. J. Effects of protein undernutrition on the dentate gyms in rats of three age groups. Brain Res. 532:271-277; 1990. 10. Dobbing, J. The later development ofthe brain and its vulnerability. In: Davis, J. A.; Dobbing, J., eds. Scientific foundations of paediatrics, 2nd ed. London: Heinmann; 1981. 11. Gage, P. D. Performance ofhippocampectomized rats in reference/ working memory tasks: Effect of preoperative and postoperative training. Physiol. Psychol. 13:235-242; 1985, 12. Goodlett, C. R.; Valentino, M. L.; Morgane, P. J.; Resnick, O. Spatial cue utilization in chronically malnourished rats: Task specific learning deficits. Dev. Psychobiol. 19:1-15; 1986.
13. Hall, R. Is hippocampal function in the adult rat impaired by early protein or protein-calorie deficiencies? Dev. Psychobiol. 16:395-411; 1983. 14. Jordan, T. C.; Cane, S. E.; Howells, K. F. Deficits in spatial memory performance induced by early undernutrition. Dev. Psychobiol. 14: 317-325; 1981. 15. Jordan, T. C.; Howells, K. F.; McNaughton, N.; Heatlie, P. L. Effects of early undernutrition on hippocampal development and function. Res. Exp. Med. 180:210-207, 1982. 16. Jordan, T. C.; Taghavi, Z. Temporal factors in spatial memory deficits induced by early undernutrition in rats. Neurosci. Lett. 21:S50; 1985. 17. La Tour, S. A.; Miniard, P. W. The misuse of repeated measures analysis in Marketing Research. J. Marketing Res. 20:45-57; 1983. 18. Morris, R. G. M. Spatial localisation does not require the presence of local cues. Learn. Motiv. 12:239-260; 1981. 19. Morris, R. G. M.; Garrud, P.; Rawlins, J. N. P.; O'Keefe, J. Place navigation impaired in rats with hippocampal lesions. Nature 297: 681-683; 1982. 20. Olton, D. S.; Walker, A.; Gage, F. Hippocampal connections and spatial discrimination. Brain Res. 139:295-308; 1978. 21. Paula-Barbosa, M. M.; Andrate, J. P.; Castedo, J. L.; Azevado, F. P.; Cameos, I.; Volk, B.; Tavares, M. A. Cell loss in the cerebellum and hippocampal formation of adult rats after long-term low-protein diet. Exp. Neurol. 103:186-193; 1989. 22. Smart, J. L. Activity and exploratory behaviour of adult offspring of undernourished mother rats. Dev. Psychobiol. 7:315-321; 1974. 23. Smart, J. L.; Dobbing, J. Increased thirst and hunger in adult rats undernourished as infants: An alternative explanation. Br. J. Nutr. 37:421-430; 1977. 24. Sokal, R. R.; Rohlf, F. J. Biometry. 2nd ed. New York: W. H. Freeman; 1981. 25. Thomas, G. J.; Brito, G. N. O.; Stein, D. P. Medial septal nucleus and delayed alternation in rats. Physiol. Psychol. 8:467-472; 1980.
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Spatial Learning Ability of Rats Undernourished During Early Postnatal Life K. S. B E D I
Department of Anatomy, University of Queensland, St. Lucia, Brisbane, Queensland, Australia, 4072 Received 16 A u g u s t 1991 BEDI, K. S. Spatial learning ability of rats undernourished during early postnatal life. PHYSIOL BEHAV 51(5) 1001-1007, 1992.--Experiments to determine whether undernutrition during early life results in deficits in spatial learning behavior has produced conflicting results. It was hypothesized that this may be due to the differing degrees of undernutrition used in the various studies, and/or to the timing of the testing procedures with respect to the period of food deprivation. These possibilities were tested by undernourishing rats between birth and 30 days of age to two different levels (i.e., level-I and level-2). The degree of undernutrition was greater at level-2 than level-l. Behavioral testing of these rats and well-fed controls was carried out in the Morris water maze when they were between either 35 and 65 or 170 and 200 days of age. Statistical analyses of the escape latency data from these experiments revealed that rats tested almost immediately after the period of undernutrition have alterations in their spatial learning behavior compared with controls. However, even a short period of nutritional rehabilitation removed any differences between control and previously undernourished groups, irrespective of the level of undernutrition. Malnutrition
Behavior
Memory
STUDIES in which the rat hippocampal formation or its connections have been lesioned have demonstrated that it is an important brain center influencing, among other things, the spatial learning capacity of the animals (l l, 19,20,25). Such lesioned animals show deficits in spatial learning capacity as demonstrated by a number of behavioral tests. Spatial learning behavior of rats is often tested using 8-, 12-, or 16-radial arm mazes [e.g. (14,15)] and the Morris water maze test (18,19). Undernutrition of rats during early postnatal life is known to cause changes in the developing brain. These alterations are not characterized by qualitatively visible lesions, but instead consist of quantitative deficits and distortions (10) of brain structure (3,4,6). A brain region particularly affected by undernutrition during early life is the hippocampal formation [e.g. (1,2,5,9)]. This observation has led to a small number of behavioral studies (5,7,12-16) designed to test the spatial learning ability of rats undernourished during early life. The results of these studies are somewhat conflicting. For example, Jordan et al. (14) examined 90-day-old rats previously undernourished during the gestation and suckling periods. The performance of these rats was significantly different from controls in a radial maze task. However, Hall (13), who used similar behavioral methods in three separate experiments, could not confirm the results obtained by Jordan et at. (14). The use of radial arm mazes to test the spatial learning ability of undernourished rats is problematical. A food reward (reinforcement) is required in order to induce the rats to perform the test. In order to accomplish this, the rats have to be hungry at the time of testing. This is normally achieved by placing the rats on a diet, slightly reduced in amount, in order to decrease the body weight of the rats just prior to testing. This is obviously a complicating factor in experiments where malnutrition or un-
1001
dernutrition is the factor under investigation in the first place. The problem is further exacerbated because there is now considerable evidence (22,23) that rats previously undernourished during early life have differential degrees of motivation to obtain rewards (particularly food or water reinforcements) compared with well-fed rats. Jordan et ai. (14) did not attempt to account for this factor. Hall (13) attempted to equate motivational levels of his nutritional groups by reducing the body weights of the controls and experimental rats to different degrees just prior to the testing procedures. However, there is no evidence presented that this maneuver was successful in equating levels of motivation. The use of the Morris water maze (18) for testing spatial learning ability in undernourished rats offers the advantage that an appetitive reward is not required. This circumvents some of the problems mentioned above. Previously published studies that have used the Morris water maze test on undernourished rats are those by Jordan and Taghavi (16), Goodlett et al. (12), Castro and Rudy (8), and Campbell and Bedi (7). Of these, the study by Goodlett et at. (12) only examined adult rats that had been undernourished from conception and throughout their lives. Castro and Rudy (8) only tested young rats, almost immediately following the period of undernutrition. The details of these studies are discussed further in this paper. The previous study carried out in my laboratory (7) examined adult rats that had been undernourished during early postnatal life but then allowed a lengthy period of nutritional rehabilitation before behavioral assessment. The length of the period of undernutrition was either 30 or 60 days and commenced at birth. No statistically significant deficits could be demonstrated in the spatial learning ability of control and experimental animals. However, the coefficients of variation between individual animals
1002
BEDI
within a given group were sufficiently large to make it uncertain whether a type It error (24) had not occurred. In the present study I have assessed whether the level of undernutrition imposed during early life could be a factor in influencing the spatial learning capacity of the animals. This paper describes the results of tests carried out on animals soon after the end of the period of undernutrition, as well as on rats given a lengthy period of nutritional rehabilitation before behavioral testing. METHOD
Animals The rats used in this study were of an outbred hooded LongEvans strain purchased from the Central Breeding Unit of Monash University, Victoria. Virgin females were housed overnight with males and the presence of a vaginal plug the next day was used to determine whether mating had occurred. Mated females were housed in separate cages. These dams were divided, at random, into three groups on the day 16 of pregnancy. The first group was designated as the control group; the second and third as the level-1 and level-2 undernourished groups, respectively. At birth all litters were standardized, by culling, to contain eight pups with at least two, but not more than four, females in each litter. There was no crossfostering of any pups between litters. The control animals and their offspring were allowed to feed ad lib throughout the experiment. The amount of food given to the level-l and level-2 dams was restricted to that shown in Table I from the day 16 of pregnancy and throughout the suckling period. All male pups were weaned on postnatal day 19 by separating them from their mothers and housing them three to a cage. Female pups were euthanized at this stage. The amount of food given to level-I and level-2 pups was restricted to the values shown in Table l until they had reached 30 days of age. The daily food intake of the level-l and level-2 rats represented about 60% and 40% of that eaten by the well-fed control animals at the same stage. Water was available to all rats ad lib throughout the experiment. Nutritional rehabilitation of the undernourished pups was commenced when they had reached 30 days of age by placing them on an ad lib diet.
Experiment 1 This was carried out on rats between 35 and 65 days of age, i.e., soon after the end of the period of undernutrition. There were 15 control and 15 level- 1 undernourished rats, each group being derived from five separate litters. There were 11 level-2 undernourished rats derived from six litters. All rats were between 35 and 65 days of age during the testing period. Because it was only possible to test about 15 rats on any given day, the available rats were subdivided into three subgroups. The first subgroup contained three control, five level- 1, and four level-2 rats. The second subgroup contained six control, four level-1, and five level-2 rats. The third subgroup contained six control, six level- 1, and two level-2 rats. The rats within a given nutritional group in these subgroups were, as far as possible, derived from separate litters. One of the subgroups was tested during each of the first 3 weeks of the test period. The order in which the individual rats were tested during a given week was randomized between nutritional groups. The testing apparatus consisted of a circular tank of diameter 1.53 m and height 0.31 m filled with water at a temperature of 25 +_ 1°C to a depth of 0. ! 8 m. The water was made opaque by the addition of about two liters of milk. The tank was located
TABLE 1 DAILY A M O U N T S (g) OF FOOD GIVEN l'O RATS D U R I N G EARLY DEVEI_.OPMENT I Indernourished Rats
Gestation G 16-birth Postnatal PND0-PND6 PND7-PND t 3 PNDI4-PNDI8 Post-weaning PND 19-PND21 PND22-PND25 PND26-PND30
Level- I
I cvel-2
12
8
17 22 27
13 18 23
3 3.5 4
2 2.5 3
G, Gestation. PND, Postnatal day. Note that during the gestation and lactation periods, it was the dam that received the food intake indicated, while after weaning it was the pups that received the food indicated(see Method Section for detailed description). in the center of a test room, the floor area measured 2.4 x 3 m, the ceiling being 2.4 m high. The walls of this room had no windows; the door leading into the adjoining room was fitted with a black curtain. A closed circuit video camera, equipped with a wide angle lens, was fitted in the center of the ceiling of the test room, directly above the pool. Other equipment placed within the test room provided distinct visual cues. The closed circuit video camera relayed a picture of the pool to a video tape recorder and monitor in the adjoining room. The image of the pool on the monitor was marked into four equal quadrants labeled N, S, E, and W. Four equally spaced start points were marked around the circumference of the pool, one for each quadrant. A 14 cm diameter platform was constructed with perspex and covered on the outside with a white colored cotton material. The height of this platform was 0.17 m so that when it was placed in the pool of water it became completely submerged to a depth of about 1 cm below the surface. The white material helped to make the platform invisible in the milky-colored water in the pool.
Spatial Learning Test--Escape Acquisition There was no pretraining period for any of the rats. The main escape acquisition test consisted of eight trials per rat per day over 3 consecutive days. Each trial consisted of placing the rat in the water at one of four starting points, such that it faced away from the center of the pool. The starting point for any given trial was chosen at random. The rats were required to swim until they found, and climbed onto the submerged platform; the time taken (escape latency) to do this for each trial being recorded. The platform was located in the same place (the approximate center of one of the quadrants) for all trials for any given rat. However, this location was randomized between rats. Once the rat had climbed onto the platform in any given trial it was left there for a period of 60 seconds before being removed to a holding cage for a further 1 to 2 minutes. After this, the rat was subjected to the next trial. All trials were recorded on the video tape recorder in the adjoining room from where the trials were monitored by the experimenter.
SPATIAL LEARNING IN UNDERNOURISHED RATS
If a rat failed to find the platform within 6 minutes of the start of any given trial the rat was removed from the pool and placed in the holding cage for a period of between 2 and 5 minutes before the next trial. Out of the 1066 separate trials in this experiment failure to reach the platform in the allotted time only occurred a total of I 1 times and involved two level-I and three level-2 undernourished rats. All 11 failures occurred during the first test day.
1003 using a repeated measures ANOVA for unequal sample sizes, and examined differences between Groups and Test Days (with all three weeks combined) as well as the interaction between them. All statistics were carried out on an IBM-compatable personal computer using a SAS statistical software package (SAS Institute Inc., Cary, NC 27512-8000) on license to the University of Queensland.
Memory Persistence Test Following the initial 3-day testing period, each rat was given one additional trial on each of days 4 and 11 of its test period. For any given rat, the platform remained in the same quadrant as that used in the escape acquisition task.
Experiment 2 This was carried out using rats between 170 and 200 days of age, i.e., after a lengthy period of nutritional rehabilitation. The testing procedures were similar to those used in Experiment 1 except as detailed below. There were ten control, nine level-1, and seven level-2 rats, each group being derived from four separate litters. As these older rats were somewhat larger and heavier than those used in Experiment 1, the spatial learning task was carried out in a larger pool with a diameter of 1.82 m and height of 0.61 m. The maximum time allowed for a rat to find the platform during any given trial was 5 minutes. Out of the 624 trials in experiment 2, failure to reach the platform in the allotted time only occurred a total of 14 times and involved four control, four level-1, and two level-2 rats. Thirteen of these failures occurred on the first test day; the remaining one occurred on the second test day and involved a control rat. In Experiment 2 the memory persistence test was carried out on day 18 as well as on days 4 and 11.
Statistics Body weight data was analyzed by two-way ANOVA procedures. Escape latency data was not normally distributed due to the fact that on occasions some rats took considerably longer to locate the platform than others, causing a degree of positive skewness. A log~otransformation (24) was, therefore, carried out on this data before further univariate analysis. (The number of replications was not considered sufficient to make multivariate analysis appropriate.) As the rats in Experiment 1 were only between 35 and 65 days of age, rats tested at the beginning of the test period could give markedly different results to those rats tested at the end of the test period. Even a few extra days of nutritional rehabilitation at this relatively young age could affect the performance of the animals; l or 2 weeks represents a large portion of the lifespan of 35-65-day-old rat. Therefore, for Experiment l the univariate analysis involved repeated measures ANOVA for unequal sample sizes with groups, weeks (i.e., weeks l, 2, or 3 during test period), days (days l, 2, or 3 during test week), and trials as the variables. This analysis used Huyhn-Feldt's adjusted probabilities necessary because of the repeated measures design of the experiment (17). In Experiment 2 all the rats were between 170 and 200 days of age; the undernourished groups having been nutritionally rehabilitated for a lengthy period of time commencing at 30 days of age. Testing such rats at the beginning or end of the test period would not be expected to greatly influence the results; 1 or 2 weeks in the life of a 200-day-old rat is a relatively short period of time. Therefore, the data in Experiment 2 was analyzed
RESULTS
Body Weights The mean body weights for the three groups of rats in both Experiments ! and 2 are presented in Table 2, together with the results of two-way ANOVA of the data. There were significant deficits in mean body weight between the three groups of rats at all ages examined. As expected, the well-fed control rats were substantially heavier than the level-t and level-2 undernourished rats. However, it was also noted that the level-1 rats were, in turn, significantly heavier than level-2 rats. Furthermore, nutritional rehabilitation after 30 days of age did not seem to restore the deficits in body weight for either level-1 or level-2 undernourished rats (Table 2).
Escape Latency Figures 1 and 2 show the mean escape latency for each trial and nutritional group for the escape acquisition task plotted on a semilog scale for Experiments 1 and 2, respectively. These show that all three groups of rats were able to learn to locate the position of the platform after a few trials. Table 3 shows the logm latency data tabulated to show group means by day and week for Experiment 1, and group means by day (with all three weeks combined) for Experiment 2. This data was subjected to an overall repeated measures ANOVA for unequal sample sizes. The results of these analyses are shown in Table 4. Examination of the data from Experiment 1 shows differences between nutritional groups during week 1, but not during weeks 2 and 3. During week 1 the undernourished rats appear to take longer to find the platform than control animals; in all cases the control rats mean logtime taken is less than that
TABLE 2 MEAN _+ SEM BODY WEIGHTS(g) OF RATS USED IN THE BEHAVIORAL TESTINGNEAR THE BEGINNING AND END OF THE TEST PERIOD
Experiment 1 Number in group Age (days) 30 60 Experiment 2 Number in group Age (days) 165 200
Control Rats
Level-1Rats
Level-2Rats
15
15
11
90.6 _+ 1.5 243.4 _+4.1
34.5 _+0.4 189.7 _+5.6
23.5 _+ 1.0 160.7 _+ 4.2
II
9
7
378.9 +_9.2 395.8 _+8.1
317.7_+6.1 336.2 _+6.9
302.9 _+ 12.1 307.0+ 13.9
Results of two-way ANOVA of above data: Experiment 1: age, p < 0.0001; nutrition, p < 0.0001; age X nutrition, p < 0.01. Experiment 2: age, p < 0.05; nutrition, p < 0.001; age X nutrition, NS.
1004
BI-~DI 10 .... ,D--- Control
........t ....... Level 1 o----
Level 2
J
0.1
I
5
I
!
10
!
15
20
25
Trial number
FIG. 1. Plots to show the mean escape latencies against a given trial number for the three groups of rats in Experiment 1. There were eight trials per day for 3 days for each rat. The effects of test week (see Method section) have been ignored for this plot. Each point represents the mean value for all the rats in a given group for a given trial number. Standard error bars are also shown. Experiment 1 was carried out on rats between 35-65 days of age. for the level- 1 and level-2 rats. This is not so, however, for weeks 2 and 3 (Table 3). These differences are reflected in the ANOVA test by a significant main effect of groups as well as a significant week X groups interaction (Table 4). In other words, the differences between the three nutritional group means vary significantly depending on whether or not the experiment was done in week 1, 2, or 3 of the test period. The mean escape latencies for all groups decreased over the three test days during any given week. These observations were reflected by a significant main effect of days and days × week interaction in the ANOVA test (Table 4). The days × group and days X week X group interactions were not statistically significant. In other words, the differences between the means for the 3 days (for all nutritional groups) varied to some extent depending on the week of the experiment. There was also a significant main effect of trials and a days x trials interaction (Table 4). All the remaining first, second, and third order interactions were not significant (Table 4). These results indicate that the intertrial variation itself varies depending on the day of the experiment, and that this intertrial variation may be significant to a greater or lesser degree, depending on the day of the experiment. This was irrespective of the nutritional group or the week of the experiment. In Experiment 2 there were no significant differences between groups, although there was significant effects of trials and days
(Table 4). The days X groups and trial X groups interactions were also not significant (Table 4).
Memory Persistence Test Table 5 shows the mean escape latencies for the groups of rats on the single trials carried out on days 4 and 11 for Experiment 1 and days 4, I 1, and 18 for Experiment 2. In Experiment 1, two-way ANOVA with the day of testing and nutrition as the two factors revealed a significant main effect of days but not nutrition. The day X nutrition interaction was also not statistically significant. These results are reflected in the observation that all groups of rats appeared to take significantly longer to locate the platform on day 11 than they did on day 4 (Table 5). The results for the memory persistence test in Experiment 2 were broadly similar to those obtained in experiment 1, and are also summarized in Table 5. G E N E R A L DISCUSSION In Experiment 1, the rats tested in weeks 2 and 3 had slightly longer periods of nutritional rehabilitation than those tested in week 1, that were in effect tested between 6 and 9 days following the end of the period of undernutrition. At the relatively young age that these rats were tested, even such small differences in age and degree of nutritional rehabilitation can be conceived to
SPATIAL LEARNING IN UNDERNOURISHED RATS
1005
10
!i
.... • ....
Control
#-
Level 1
o---
Level 2
1
0.1
I
0
5
I
10
I
|
15
20
25
Trial number
FIG. 2. Plots to show the mean escape latencies against a given trial number for the three groups of rats in Experiment 2. There were eight trials per day for 3 days for each rat. The effectsof test week (see Method Section) have been ignored for this plot. Each point represents the mean value for all the rats in a given group for a given trial number. Standard error bars are also shown. Experiment 2 was carried out on rats between 170-200 days of age. markedly effect their performance in behavioral tasks. The results of this experiment showed significant intergroup differences and a significant week × group interaction. In all cases, the mean log,o latency for the controls was less than that for the level-1 and level-2 rats during week 1. However, by weeks 2 and 3 the differences were not so obvious and were not significant, although in all cases the level-2 rats took marginally longer to find the platform. These results indicate that even a short period of nutritional rehabilitation can remove any differences in spatial learning capacity between nutritional groups. This finding was further confirmed in Experiment 2, where the rats were given a lengthy period of nutritional rehabilitation before being tested in the Morris water maze. The results of this experiment showed that there were no statistically significant long term effects on the performance of these rats, compared with age-matched controls, in the spatial learning task used. This finding is in agreement with our previous research (7). Our results indicate that all groups of rats were capable of learning to find the hidden platform over the 3-day test period of the experiment, usually by the end of the first day. Having learned to find the platform, little or no further improvement would be expected on days 2 and 3. This, in fact, seems to be the case for most groups of rats in both Experiments 1 and 2. The memory persistence tests revealed that the rats took slightly but significantly longer to locate the platform on day 11
than on day 4 in Experiment 1 and on days 11 and 18 than on day 4 in Experiment 2. Whether this indicates a marginal loss of memory between these days is not of great interest. What is important is that both control and undernourished rats appeared to behave in the same way in this respect. It should be mentioned that proximal-cue navigation using a visible platform was not tested in the present experiments. In previous work using rats given a lengthy period of nutritional rehabilitation following undernutrition during early postnatal life we (7) could not find any differences in proximal-cue navigation between control and experimental rats. The experiments in this previous work were of a similar design to that in Experiment 2 in the present study. It was, therefore, felt to be unnecessary to repeat the proximal-cue navigation test in Experiment 2. It would, however, have been useful to test proximal-cue navigation in rats soon after the period of undernutrition (i.e., those in Experiment 1). Such a test may have served as a kind of pretraining period insofar as the rats could have learned that escape was possible by finding a platform, making interpretation of the results more difficult. Unfortunately, the number of rats available in the present study precluded the possibility of carrying out the proximal-cue navigation test on a separate subgroup of rats than those used in the main escape acquisition test. It was considered best to concentrate all the available animals into the crucial distal-cue navigation test, thereby increasing the likeli-
1006
BEDI TABLE 3 GROUP MEANS OF Iog~o LATENCY BY DAY AND WEEK Day 1 Experiment 1 Week 1 Control [24] Level- 1 [40] Level-2 [32] Week 2 Control [48] Level- 1 [32] Level-2 [40] Week 3 Control [48] Level- 1 [48] Level-2 [ 16] Experiment 2* Control [80] Level- 1 [72] Level-2 [56]
Day 2
Day 3
1.103 1.709 1.680
0.862 1.357 1. I 13
0.720 0.926 1.007
1.162 1.124 1.459
1.033 0.994 1.055
0.832 0.855 0.981
1.315 1.228 1.401
0.930 0.926 1.108
0.849 0.914 1.061
1.598 1.495 1.573
1.151 1.249 1.225
0.963 1.038 1.067
Number of trials are in brackets. * In this experiment the data was analyzed with weeks 1, 2, and 3 combined.
hood of detecting any differences in escape latencies between nutritional groups, had they existed. Spatial learning ability of undernourished rats has been previously investigated using the Morris water maze (7,8,12,16). Goodlett et al.'s (12) experiments were designed to examine adult rats that had been undernourished from conception and throughout their lives. They concluded that chronic malnutrition in rats did not impair spatial localization ability and spatial memory processes. However, the malnourished rats had a somewhat slower rate of learning than controls. The results of Goodlett et al.'s (12) experiment are not directly comparable with our present work. Long term and ongoing malnutrition during the test period, like that used by Goodlett et al. (12), may well result in different behavioral deficits than those induced by a short period of undernutrition during early life. The study by Castro and Rudy (8) is more directly comparable to the present investigations. They examined 22- to 30-day-old rats that had been undernourished between postnatal days 2 and 18. In their Morris maze tests they employed either a visible platform (proximal-cue navigation) protruding just above water level or a hidden platform (distal-cue navigation) the surface of which was just below the level of the opaque water in the pool. They concluded that proximal-cue navigation was not influenced by nutrition. This finding was confirmed by us (7) in previously reported experiments. Castro and Rudy (8) also found that malnutrition only had a mild influence on distal-cue navigation (i.e., hidden platform test) at 30 days of age. However, 22-27day-old rats had significantly worse distal-cue navigation than control animals. In Experiment l with the hidden platform, rats were also tested fairly soon after a period ofundernutrition during early life. As mentioned above, the results indicate that there are initial differences between nutritional groups in the performance in the Morris water maze. These findings, therefore, go to support the observations made by Castro and Rudy (8). However, it can be argued that the performance of rats tested soon after a period of undernutrition could be influenced by their physical weakness rather than by, or in addition to, any
TABLE 4 OVERALL REPEATED MEASURES UNIVARIATE ANOVA ()t:: log~o Source Experiment 1 Group Week Week × group Error Days Days X group Days X week Days X week × group Error (days) Trial Trial X group Trial X week Trial X week x group Error (trials) Days x trial Days × trial X group Days × trial X week Days × trial X week X group Error (days x trials) Experiment 2 Groups Error Days Days X groups Error (days) Trials Trials X groups Error (trials) Days X trials Days X trials x groups Error (days X trials)
d/
MS
t
,
2 ~ 4 32 2 4 4 8 64 7 14 14 28 224 14 28 28 56 448
3.676 0.956 1.701 0.359 15.324 0.223 0.565 0.280 0.212 3.539 0.099 0.077 0.082 0.096 0.699 0.010 0.085 0.072 0.101
10.25 2.67 4.75
0.0004 0.0849 0.0040
72.36 i.05 2.67 1.32
0.0001 0.3821 0.0492 0.2586
37.06 1.03 0.80 0.86
0.0001 0.4206 0.6655 0.6707
6.96 0.99 0.84 0.72
0.0001 0.4749 0.6973 0.9344
2 23 2 4 46 7 14 161 14 28 322
0.129 1.001 14.895 0.242 0.427 2.091 0.131 0.170 0.287 0.120 0.134
0.13
0.8812
34.93 0.57
0.0001 0.6859
12.28 0.77
0.0001 0.6815
2.14 0.89
0.0126 0,6169
changes in the functioning of the central nervous system. The results of the present study supports this possibility. Any deficits in spatial learning capacity disappeared with progressively longer periods of nutritional rehabilitation. In other words, in Experiment 1, group differences were only observed during week 1 of
TABLE 5 MEAN 4-_ SEM ESCAPE LATENCY (SECONDS) OF RATS IN TRIALS Control Experiment 1 Number in group Day4 Day 11 Experiment 2 Number in group Day4 Day ll Day 18
Level-1
Level-2
15 15 11 6.43_+ 1 . 1 4 8.69_+ 1 . 1 2 11.34_+2.34 22.3 -+ 7.4 23.1 -+ 10.3 21.4 _+7.2 10 13.4 _+ 3.8 19.7 _+ 7.7 30.4 _+ 10.3
9 14.1 -+ 2.3 26.7 _+11.4 16.9 _+ 5.0
7 14.0 _+4.5 23.0 _+7.1 21.7 _+4.9
Results of two-way ANOVA of above data: Experiment 1: day, p < 0.05; nutrition, NS; day × nutrition, NS. Experiment 2: day, NS; nutrition, NS; day x nutrition, NS.
SPATIAL L E A R N I N G IN U N D E R N O U R I S H E D RATS
the test and not during weeks 2 and 3. The results of Experiment 2, which tested rats following a lengthy period of nutritional rehabilitation, showed no intergroup differences. Taken together, these observations supports the hypothesis that the group differences observed in Experiment 1, and those described by Castro and Rudy (8) were transient and not long-term effects of the period of undernutrition. The only studies to have examined previously undernourished rats following a lengthy period of nutritionally rehabilitation with the Morris water maze test were those by Jordan and Taghavi (16) and that carried out in my laboratory (7). In our previous work (7) that employed both a visible and a hidden platform test, we could not demonstrate any statistically significant effect of undernutrition during early life on spatial memory performance. The study by Jordan and Taghavi (16) reported in a short abstract (and, therefore, necessarily devoid of detailed statistical information) found that the persistence of spatial memory of previously undernourished rats was significantly poorer than
1007 well-fed controls. There is no immediately obvious explanation for the discrepancy of this finding with our own present and previous work (7). In conclusion, the present experiments provide evidence that rats tested almost immediately after a period of undernutrition during early life have deficits in their spatial learning behavior compared with well-fed controls. However, there was strong evidence to indicate that nutritional rehabilitation, even for a period as short as 2 or 3 weeks, was capable of removing such deficits. In other words, undernutrition during early postnatal life did not permanently affect the spatial learning behavior of rats, as tested with the Morris water maze test. ACKNOWLEDGEMENTS 1 would like to thank Mr. D. Murray for providing research assistance during this work and Dr. Joan Hendrikz for providing advice and help with the statistical analysis. This research was funded by a grant from the NH and MRC of Australia.
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