spatial learning and memory in aging c57bl/6 mice

NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 23, NO. 2

SPATIAL LEARNING AND MEMORY IN AGING C57BL/6 MICE

Craig Weiss, Arati Shroff and John F. Disterhoft Northwestern University Medical School, Department of Cell and Molecular Biology

Chicago, IL 60611

(Accepted JziZy 1, 1998)

SUMMARY We evaluated spatial behaviors as a function of age in C57BL/6 mice (4, 16, and 24

months). Simple exploration was measured in an open field, spatial memory was assessed with spontaneous alternation in a Y maze, and spatial learning and memory were measured using a 16 well hole-board with water rewards. The results indicate significant age-related decreases in exploration, significantly less alternation in the Y maze for the two older age groups, and significant age-related impairments in reference and working memory with the hole-board task. Deficits in the hole-board were not due to a lack of exploration, or time to complete the task. A factor analysis indicated that the behavioral measures factor differently with each other as a function of age, and a step-wise regression of the behavioral measures with age indicated that each task uniquely and independently revealed age-related impairments. In conclusion, the three tasks reported here can form a battery of tests to examine changes in spatially mediated behaviors and in the molecular/physiological mechanisms within the limbic system of the aging brain.

Key Words: maz e

hippocampus, hole-board, open field, spontaneous alternation, water maze, Y

INTRODUCTION

Spatial learning paradigms have provided considerable data for understanding the

mechanisms underlying learning and memory in rodents. These paradigms have been

useful for developing model systems for the analysis of the effects of aging on learning and

0 1998 Wiley-Liss, Inc.

78 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 23, NO. 2

memory. These model systems can then be used to understand information processing

through the hippocampus and prefrontal cortex in normal and abnormal human aging

(13) . Mice in particular need to be studied in a variety of tasks since they have been

reported to behave differently than rats in spatial tasks (22,38,39) and are being widely used

for the study of spatial behavior following the addition or deletion of single genes

(1,2,15,16,29,40,41). This study is part of our multi-faceted characterization of age-related

learning deficits in rodents. This particular experiment was performed to test the

hypothesis that different spatial tasks can be selected which utilize different memory

functions and which are

to provide measures of

differentially affected by the aging process. The

exploration in an open field (17), spontaneous

tasks were selected

alternation in a Y

maze (ll), and learning and memory in an appetitively motivated 16-well hole-board

(25,28,33,34,35,37).

The open field maze was included since it relates to spatially oriented behavior and

has been used with success by Hsiao et al. (17) to discriminate mice that overexpress the

gene for the amyloid precursor protein (APP) from their normal litter mates. Normal

mice explore the cage. Transgenic mice affected by the overexpression of the APP gene

tended to stay within the center of the cage and exhibit neophobia.

The Y maze assesses spontaneous alternation which is a natural, hippocampally-

dependent, behavior in young rodents (11). Older mice lose this alternation behavior (21),

especially if a one minute intertrial interval is imposed (30). This maze was included

because it is a simple test of working memory. There are also minimal physical demands

that can be affected by aging.

The hole-board was included to examine changes in working and reference spatial

memory in a land based maze, i.e., memories based upon the use of extramaze cues. The

hole-board typically requires learning several food reward locations within a matrix of

many possible locations. This task is considered a spatial learning paradigm since after

repeated trials animals learn to avoid empty holes and visit only those locations baited

with food (25). Odor recognition is eliminated by baiting the false bottoms of the “empty”

NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 23, NO. 2 79

holes. This manipulation encourages the subject to use extra-maze cues for spatial

orientation within the hole-board arena.

This task is similar to the water maze (7,8,23,24) in that extra-maze cues are used to

solve the task, and both working and reference memory ratios can be calculated. However,

the hole-board provides an appetitively motivated, land based maze that avoids olfactory

cues. The hole-board is also easily manipulated to change the memory load placed upon

the subjects.

METHODS Subjects Male C57B1/6 mice were obtained from Charles River Laboratories via a contract

with the National Institute of Aging. Mice at 4 months, 16 months, and 24 months of age were tested. There were six mice tested in each age group. The mice were maintained on a 14/10 hr light/dark cycle with lights on at 0700. The mice were generally tested between 1000 and 1500 hours. The mice had free access to food, and were maintained at about 90% of their starting weight by placing them on a water restricted diet, i.e., they only received water for 30 minutes per day (during and immediately after the hole-board testing session). All subjects were treated in accordance with NIH policies and with a protocol approved by the animal care and use committee of Northwestern University. Each animal was tested in all three of the following tasks on each day, and in the same order, i.e., open field, then Y maze, then hole-board. The first four days in the hole-board were used for habituation purposes so that there are 10 data points for the hole-board, and 14 data points for the other tasks.

Apparatus and Procedure Onen Field The open field consisted of a standard plastic animal cage that measured

24 cm x 14 cm x 12 cm tall. A thin fresh layer of bedding was scattered across the floor of the cage prior to testing each subject. Each mouse was taken from its home cage and placed within the center of the test cage. The number of corners that were explored within 30 set (17) was recorded for each of 14 days to keep the sequence of testing the same throughout the duration of testing with the hole-board.

Y Maze The Y maze was constructed according to the specifications of Stone, Rudd and Gold (30). Each of the three arms was 60 cm in length, 3.5 cm wide at the bottom and 14 cm wide at the top. Each mouse was placed within the “home” arm and then released to choose one of the other arms. It was then trapped within the new arm for 30 sec. The mouse was then placed back within the home arm for 30 set and then released to choose one of the other arms again. The mouse was returned to its home cage immediately after the second choice was made. If the second choice was different from the first choice the mouse was scored as alternating. A mean percent alternation was calculated for each mouse at the end of 14 days.

Hole-Board The hole-board measured 70 cm x 70 cm x 40 cm and contained 16 equidistant holes (13 cm apart, 3.5 cm diameter). Each side of the hole-board contained a


start box that projected out from the arena and which measured 21 cm x 11 cm x 11 cm. This box had a hinged top that was opened to place the mouse inside. The box also had a guillotine door which controlled access to the hole-board arena. The maze was surrounded by fixed extra maze cues which also extended above the maze to the ceiling (160 cm from top of maze). One wall had a large window, another had filing cabinets and hanging bookshelves, another had a door, and a fourth wall had more cabinets and bookshelves.

Each of four daily trials began by placing a mouse in one of the four randomly selected start boxes. The guillotine door was raised after 10 s and lowered after the mouse entered the arena. A visit to a hole was scored when the mouse dipped its head within a hole. A record was kept of the sequence of holes explored, and the total number of explored holes was recorded as well as the time taken to find all four rewards, i.e., completion time. Each trial ended when all four rewards had been taken, or after three minutes had elapsed. At the end of a trial the mouse was removed from the arena and placed back within a different randomly selected start box to encourage the use of extramaze cues. The mouse was placed back in its home cage after the last trial of the day.

The experiment began with four days of habituation to the hole-board. All 16 holes were baited with a 30 ~1 drop of distilled water during these sessions, and each trial was separated by 30-40 s. The arena was cleaned with 5% ammonium hydroxide between each trial. After the habituation trials the search patterns for each mouse were recorded while they searched for the four baited holes out of the 16 possibilities. This was repeated for each of the following ten days. The same four randomly chosen holes were consistently baited for each mouse for the remainder of the experiment, but each mouse had its own randomly selected set of baited holes. The other 12 holes were empty.

The reference memory ratio (RMR), and working memory ratio (WMR) were calculated according to the method of Van der Zee et al. (33). The RMR was calculated as the number of visits to the correct set of holes (total baited hole visits and baited hole revisits) divided by the total number of hole visits (baited and non-baited holes). The WMR was calculated as the number of visits to baited holes while baited (this was often 4) divided by the total number of hole visits.

The data are presented as means + SE. All of the data were analyzed by analysis of - variance (ANOVA). Age served as a between group factor, and data from successive days of testing were used as a repeated measure factor. Data from the Y maze were averaged to yield one score per subject that was analyzed by ANOVA without repeated measures. An ANOVA that resulted in a p value of 0.05 or less was deemed significant. Post hoc analyses were done using the least significant difference (LSD) test. Analyses were performed with the StatVIEW 4.0 software package (Abacus Concepts, Berkeley, CA, USA).

RESULTS ODen Field The average number of corners explored for each age group is shown in

Figure 1. The ANOVA indicated that there was a significant effect of age (F2,15=85.6,

p<O.OOOl), a significant effect of days (F13,195=11.1, pcO.0001) and a significant interaction of - age and days of testing (F26,195=1.65, p=O.O3).


12 1 -

8-

6-

4- :

2 - +- 04m

_ -16m

4 24m n-4 ”

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 1. The mean number of corners (&SE) explored within 30 s are plotted as a function of age and 14 days of testing. Note that the older mice explore fewer corners than the younger mice, but that they still explore more than one corner per session (cf. ref. 17).

Post-hoc tests indicated that overall both the 16 and 24 -month-old mice explored

significantly fewer corners than the 4 -month-old mice (p<O.OOOl), and the 24-month-old

mice explored significantly fewer corners than the 16-month-old mice (p=O.O5). The 4 -

month-old mice had the largest change in exploration over the course of 14 days. The 4 -

month-old mice explored a mean of 10.1 corners on the first day, a mean of 6.7 corners on

the last day, and a mean of 8.2 + 0.19 overall. The 16 -month-old mice explored a mean of

8.3 corners on the first day, a mean of 5.0 corners on the last day, and a mean of 5.7 + 0.17

overall. The 24 -month-old mice explored a mean of 7.0 corners on the first day, a mean of

4.2 corners on the last day, and a mean of 5.2 L- 0.14 overall.

Y maze The percent of trials with alternation behavior in the Y maze was calculated

for each mouse at the end of 14 days of testing. A value of 100 indicates alternation on

every day of testing, 50 indicates alternation at a chance level, and values less than 50

indicate a tendency to return to the first selected arm, i.e., perseveration rather than

alternation. The mean percent alternation for the three ages are shown in Figure 2.


Mean Percent of Spontaneous Alternation Figure 2. A graph of the 100 1 I I I I behavior for each age groun

I Alternation in the Y maze. ” V\lues

809

60-

40-

20-

0-L l&l Age

24m

c I P I , Random

: I

I

- Perseveration

represent the mean (&SE) percent of days on which subjects exhibited spontaneous alternation. 100% indicates alternation 0 n every day of testing, 50% indicates a random preference for the two arms, 0% indicates that the first chosen arm was reentered.

The 4- month-old mice had a value of 81 & 4. The 16- month-old mice had a value

of 52 k 8. The 24- month-old mice had a value of 37 + 7. There was a significant difference

for the percent alternation among the age groups (F2,15=12.3, p=O.O007). Post-hoc tests

indicated that the 4- month-old mice alternated significantly more than either the 16- or

24- month-old mice, and the 16- and 24-month-old groups were not significantly different

from each other.

Hole-Board The results for the reference memory ratio and the working memory

ratio are graphically summarized in Fig. 3A and 3B. An ANOVA for each ratio indicated

similar results. There was a significant interaction of age and days for the mean RMR (F

18,135=13.4, p<O.OOOl) and the mean WMR (F 18,135=16.1, pcO.OOOl), as well as significant

main effects of age and days for both ratios (age RMR: F&15=29.8, age WMR: F&15=77.9, days

RMR: F9,135=96.4, days WMR: F9,135=95.9, all p values ~0.0001).

Post-hoc tests on the RMR indicated that all three age groups were significantly

different from one another (Fig. 3A). The 4 -month-old mice had a mean RMR of 0.49 ,+

0.02. The ratio increased from 0.30 + 0.01 on day 1 to 0.84 & 0.02 on day 10. The 16 -month-


old mice had a mean RMR of 0.42 & 0.01. The ratio increased from 0.29 + 0.02 on day 1 to

0.51& 0.02 on day 10. The 24 -month-old mice had a mean RMR of 0.34 & 0.01. The ratio

increased from 0.29 ,+ 0.02 on dav 1 to 0.51+ 0.02 on dav 10.

Reference Memory Ratio

t V.” . . . I w . . . . .

12 3 4 5 6 7 8 9 10

Day

C. Mean Completion Time (s) D. Number of Explored Holes per Day 180

160

140

120

100

80

60

40

20 n

+ 04m

L 16m

” - - - - - - - - - -

12 3 4 5 6 7 8 9 10

Day

1.0

0.8

0.6

0.4

0.2

0.0

100 90

80 70

60 50 40

30 20

10 0

B. Working Memory Ratio

1 + 04m t

1 I . . . . . . . . I I

12 3 4 5 6 7 8 9 10

Day

-T- 24m F

12 3 4 5 6 7 8 9 10

Day

Figure 3. Graphs of the mean memory ratios (&SE) for the 10 days of training in the hole-board. A. The reference memory ratio represents how often the mice return to a well that was within their baited set. B. The working memory ratio represents how well the mice return to the set of baited wells that still have rewards in them for any given trial. C. A graph of the mean time required for the mice to find all four rewards. A maximum of 180 s was permitted for each subject. Notice that the oldest mice were as fast as the young mice after several days of training. D. A graph of the mean total number of holes explored per day, i.e., the sum of all four trials.

Post-hoc LSD tests on the WMR indicated that all three age groups were significantly

different from one another (Fig. 3B). The 4 -month-old mice had a mean WMR of 0.45 +


0.02. The ratio increased from 0.22 + 0.01 on day 1 to 0.76 + 0.04 on day 10. The 16 -month-

old mice had a mean WMR of 0.37 ,+ 0.01. The ratio increased from 0.22 ,+ 0.06 on day 1 to

0.45 & 0.02 on day 10. The 24 -month-old mice had a mean WMR of 0.24 + 0.01. The ratio

increased from 0.20 + 0.01 on day 1 to 0.37 + 0.01 on day 10.

Learning of the hole-board task was also indicated by decreases in the time that it

took the mice to locate the four baited holes, i.e., to complete a trial. The results for each

age group are indicated in Figure 3C. An ANOVA for these data indicated that there was a

significant effect of age (F&15=25.4, p<O.OOOl), a significant change over days (F9,135=44.1, - p<O.OOOl), and a significant interaction of age and days (F18,135=4.9, p<O.OOOl).

The interaction is due to the rapid decrease in the completion time for the 4-

month-old mice

mice (days 4-9).

from each other. The 4 -month-old mice had a mean completion time of 49.3 + 4.9 s. The

A L

and the temporary increase in completion time for the 24- month-old

Post-hoc tests indicated that each age group was significantly different

16 -month-old mice had a mean completion time of 65.5 + 4.3 s. The 24 -month-old mice

had a mean completion time of 87.9 + 4.0 s. The significant change over days was due to a

decrease from 124.5 + 6.9 s on day 1 to 22.7 + 1.7 s on day 10. The completion time for the 4-

month-old mice decreased from 131.0 + 13.3 s on day 1 to 14.7 + 1.0 s on day 10. The time

for the 16 -month-old mice decreased from 121.4 + 13.0 s on day 1 to 26.4 + 2.3 s on day 10.

The time for the 24 -month-old mice decreased from 121.2 & 11.6 s on day 1 to 27.0 + 2.0 s on

day 10. It is important to note that all of the mice completed the trial within the allotted

time, and that by the last day there was little difference among the three age groups in the

time required to complete a trial.

The RMR for the four days of habituation was also calculated based upon

exploration of the wells that were to be baited. An ANOVA indicated that there were no

significant differences among the age groups (F2,15= 0.9) and no significant interaction of

age and days of habituation (F6,45= 1.06). Those results indicate that there was no prior

preference for the set of holes to be baited.


The last variable to be described is the total number of holes explored for each day.

The mean values for each age are shown in Figure 3D. An ANOVA indicated a significant

effect of age (F2,15=95.8, p<O.OOOl), days (F9,135=61.7, pcO.OOOl), and a significant interaction

of the two effects (F18,135=9.02, pcO.0001). Post-hoc tests indicated that each age group was

different from the others. The 4 -month-old mice had a mean of 42.4 + 2.2 visits per day.

The 16 -month-old mice had a mean of 49.0 + 1.9 visits per day. The 24 -month-old mice

had a mean of 71.5 + 2.1 visits per day. The number of visits decreased over days from a

high of 75.8 + 3.0 on day 1 to a low of 34.8 ,+ 2.6 on day 10. The greatest change was in the 4 -

month-old mice which decreased from a high of 76.5 + 6.8 on day 1 to a low of 21.0 + 0.7 on

day 10. The 16 -month-old mice decreased their number of visits from a high of 74.3 + 4.6

on day 1 to a low of 38.0 + 2.2 on day 10. The 24 -month-old mice decreased their number

of visits from a high of 76.5 & 5.0 on day 1 to a low of 45.3 & 1.6 on day 10.

The number of visits during the habituation sessions were also analyzed. There

were significant differences among the age groups due to the greater number of visits by

the 4 -month-old mice, but by the last day of habituation, there was no difference among

the age groups, i.e., all of the age groups explored a similar number of holes. An example

of the search behavior on day 7 of training for a typical mouse from each age group is

shown in Figure 4. This day was selected since the RMR and WMR exhibited large

differences among the ages.

Correlations of Behavior and Age- There were six variables from the three

behavioral tasks that were: 1) correlated with age, 2) intercorrelated for each age group

using a factor analysis, and 3) assessed for differential changes with age using a stepwise

regression. The variables were the mean corner index (CI), mean percent alternation (Alt),

mean RMR, mean WMR, mean number of visits in the hole-board (Vis), and the mean

completion time (Time) for the hole-board. The means were calculated for each subject

over the 10 days of testing.

Correlations with age were determined with a multiple regression. This indicated

that all of the variables were significantly correlated with age. The r2 values for the simple

correlations with age were: CI 0.86, WMR 0.86, RMR 0.79, Vis 0.77, Time 0.74 and Alt 0.62.


4 month old 16 month old \

b 28s

24 month old r(

Figure 4. Examples of the search patterns taken by a typical mouse from each age group to find 4 rewards out of a matrix of 16 locations. The locations of baited wells are shaded. The solid circles indicate an explored hole. Solid circles outside of the arena indicate the location of the start box. Data from the first and last trial of day seven are shown. The time taken to find all four rewards for each of these trials is also indicated.

The factor analysis was performed separately for each age group. An analysis of the

4-month old mice indicated that the six variables clustered into three factors with

eigenvalues >l. The first factor included RMR, WMR, and Vis. The correlations (factor

loadings) were 0.98,0.93, and -0.79 respectively. The second factor included CI and Time

with correlations of 0.94 and 0.84 respectively. The third factor only included Alt which

had a correlation of 0.96. Factors one through three respectively accounted for 43.6%,

33.6% and 20.8% of the variance in the data. Thus, the data set for 4- month old mice

could be best summarized by using measures which came from the three different tasks,

i.e., hole-board (RMR or WMR), open field (CI), and Y maze (Alt).

NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 23, NO. 2 - 87

Data from the 16-month old mice segregated into only two factors with eigenvalues

>l. The first factor included RMR, WMR, Vis and Time. The correlations for these

measures ranged from 0.935 to 0.995. The second factor included both CI and Alt with

correlations of 0.94 and -0.93 respectively. These two factors accounted for 64.0% and 29.7%

of the variance respectively. Thus, in comparison to 4-month old mice, the variable Time

moved from factor 2 to factor 1, and the alternation score moved from factor 3 to factor 2.

Thus, the data set for the 16-month mice could be best summarized by using the hole-

board (RMR, WMR, Vis or Time) and either the open field (CI) or the Y maze (Alt).

Data from the 24-month old mice also segregated into three factors, but these factors

were different than those of the 4-month old mice. The first factor included Vis, Alt, and

WMR. The correlations were 0.86, 0.85, and 0.78 respectively. The second factor only

included CI which had a correlation of 0.90. The third factor also included only one

variable, i.e., RMR. That measure had a correlation of 0.86. Factors one through three

respectively accounted for 43.7%, 31.2% and 23.0% of the variance in the data. Thus, this

set of data could be adequately summarized by using measures from either the hole-board

(Vis) or Y maze (Alt), and the open field (CI). The RMR from the hole-board also adds

significant information for the 24- month-old mice that is independent of the other

measures, including WMR.

The last correlational analysis was a stepwise regression to determine how each

measure was affected by age. The measure with the greatest partial correlation was the

mean CI (partial r= -0.93); it entered the regression with an F value of 102.3 (pcO.0001). The

second variable to enter the regression was the mean WMR (partial r= -0.80). This variable

had an F value of 26.7 (pcO.0001). These two measures yielded an adjusted r2 of 0.945. The

third variable entered the regression with a p value >0.05. This was examined to

determine if the third task was also significantly affected by age. The third variable was the

mean Alt (partial r= -0.42). It entered the regression with an F value of 2.9 (p=O.ll) and

raised the adjusted r2 to 0.96.

The results of the stepwise regression were then used to generate an equation to

calculate a new measure we termed “cognitive age”.

The equation is: CogAge = -3.04 CI -38.86WMR - 5.OAlt + 50.56.


Since the coefficients of the terms in the equation used to calculate the cognitive age

can not be compared directly due to the measures having different scales, the standardized

coefficients are more informative. These coefficients were -0.499, -0.416, and -0.141 for CI,

WMR, and Alt respectively.

DISCUSSION The experiments described here used the hole-board as well as an open field and Y

maze to evaluate the effects of age on spatial behavior and spatial learning and memory in

the same animals. The three tasks were used to examine different aspects of spatial

behavior, and each task was found to exhibit independent age-related impairments. The

correlations of spatial behavior measures from the different tasks with age suggest that the

behaviors are differentially affected, and are not likely to be using identical memory

processes. For example, the youngest mice were most different from the others in the

open field and the oldest mice were most different from the others in the hole-board.

The regression analysis also indicated that each task was significantly affected by age.

Surprisingly, the corner index from the simple open field task was most affected. The

working memory ratio was only slightly less affected as indicated by the standardized

coefficients from the regression analysis. Other measures from the hole-board (RMR,

number of holes visited, and completion time) were also affected by age, but the values of

the previous measures were sufficient to explain the effect of age upon the subject.

Lamberty and Gower (21) performed a similar analysis on data from the water maze, plus

maze and Y maze, and also found an independence of age-related behavioral changes.

Exploration in the open field was used to assess exploratory behavior since Hsiao et

al. (17) used this task as a behavioral assay for the detection of mice with the amyloid

precursor protein (API?) transgene. Although our mice exhibited an age-dependent

decrease in the number of corners explored, the oldest mice still explored many more

corners than the number reported for API? transgenic mice by Hsiao et al. (17) in FVB/N

mice. Their affected mice scored zero on at least three consecutive days. All of our mice,

including the oldest, always explored more than four corners in the allocated time. These

results suggest that Alzheimer-like pathology is associated with more severe changes in

open field behavior than that associated with normal aging.


The Y maze was used to examine spontaneous alternation (SA). This task does not

involve any reward and is a simple test of working memory. Stone, Rudd and Gold (30)

used this task with a one minute intertrial interval and reported a decrease in the percent

SA with age, i.e., old rats alternated at chance levels while young rats alternated at about

70%. We also found an age-related decrease in SA which was stable over two weeks of

testing. The young mice had a mean SA score of 81%. This score is relatively high (17-

19,30) and may be due to our limiting the mice to two choices per day (6). Relative to

young mice, our middle aged (16 m) and aging mice (24 m) alternated at chance levels. A

change in SA behavior with age was also made evident by the factor analysis, i.e., at four

months of age SA was in the third factor and accounted for 21% of the variance, at 16

months SA was part of the second factor and accounted for 30% of the variance, and at 24

months of age SA was part of the first factor and accounted for 44% of the variance. Thus,

the Y maze becomes more informative of cognitive deficits at older ages.

Lastly, the hole-board was used to determine if the effects of aging on spatial

learning are similar to the effects of hippocampal lesions (25,26,37) and aging (32) in the rat.

Our results indicate that aging mice show impairments that are similar to those created by

hippocampal lesions in the rat prior to training (25,26), or after training (37). The results

are also in general agreement with the age-related impairment in the rat reported by Van

der Staay et al. (32). However, direct comparisons cannot be made because of

methodological differences and differences in the lifespans of the two species.

The hole-board was chosen as a land based alternative to the water maze

(3,7,8,12,23,24,31), which has been widely used to evaluate the effects of age and limbic

lesions on spatial memory (3,12,23,24,31), since mice have been reported to perform better

under such conditions (38,39). The hole-board offers the opportunity to easily change the

memory load and demand of the animal, i.e., different ratios of baited to nonbaited holes

can be used, and it uses a natural appetitive foraging behavior. This natural foraging

behavior is very apparent while watching the mice perform the task, i.e., the mice do not

return to the center of the maze after exploring each hole (see Fig. 4). The behavior of the

young mice is especially impressive. They quickly proceed to the correct area of the maze,

regardless of their starting point, and then initiate a consistent and accurate search pattern.

This is indicated by the increase in the RMR and WMR, and the decrease in the


completion time. The aged mice locate the correct areas more slowly and appear to have

more trouble finding all four rewards. This was revealed by the separation of the RMR

and WMR into different factors for the old mice, and is indicated by the greater total

number of holes explored and the greater completion time. This age-related deficit is only

partially reduced with training. The aged mice do however find all four rewards well

within the limits of the allocated time which suggests that they are highly motivated to

find the water rewards.

Results similar to the changes in the RMR for our young mice were reported by Van

der Zee et al. (33) in a similar, but food rewarded task. They found a learning-related

increase in immunoreactivity to the gamma isoform of protein kinase C (KC) in

hippocampal soma and dendrites. They then repeated the experiment (34) and found a

learning-related increase in immunoreactivity for muscarinic acetylcholine receptors in

hippocampal pyramidal cells of area CAl-CA2, and a learning related decrease in the

immunoreactivity of the nonpyramidal neurons of the same region. Increases i n

hippocampal, but not cortical, KC activity were also reported by Wehner et al. (36) to

correlate with performance in the water maze. These studies indicate that the

hippocampus is involved in mediating spatial learning in the mouse, and that the hole-

board task is appropriate to study age-related and learning-related changes in the

hippocampus.

In conclusion, the three tasks reported here can form part of a battery of tests to

examine changes in spatially mediated behaviors and in the molecular/physiological

mechanisms within the limbic system of the aging brain. This is especially important

since recent evidence indicates that neuronal numbers in the hippocampus are preserved,

and that age-related learning deficits in the water maze can not be accounted for by

neuronal loss (27) or decreases in cholinergic input to the hippocampus (4) or

hippocampus and cortex combined (5). However, the number of axospinous junctions in

the hippocampal dentate gyrus do decrease with age in the rat (14). This suggests that

physiological, synaptic, or molecular mechanisms, such as calcium regulation (9,lO) change

with age and could account for the age-related learning impairments. The battery of tasks

we describe here may be used as an alternative, or an adjunct, to the more commonly used


water maze task, and may be used to evaluate the behavioral consequences of cellular and

molecular changes among several strains of mice in a comprehensive fashion.

This research was supported by the Alzheimer’s Association PRG94054, Illinois Dept. of Public Health and NIH AG08796. We thank Dr. Eddy van der Zee, Dr. Alfred Rademaker, and Dr. Sara Solla for their assistance.

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spatial learning and memory in aging c57bl/6 mice

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