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National Food Research Institute, Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • age • docosahexaenoic acid • phosphatidylcholine • maze-learning ability • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Barnes 1979
). The results from an early study suggested
that the concentration of HUFA in total phospholipids from the frontal
brain cortex was reduced with increasing age (Bowen et al. 1973
). A decrease in HUFA levels, particularly DHA and
arachidonic acid [20:4 (n-6)], in total brain lipids of aged rats has
also been reported (Suzuki et al. 1989
). Thus, it has
been proposed that changes in the fatty acid composition and the
metabolism of brain lipids occur during aging, and these appear to be
correlated in part with an age-related deterioration of functions
of the central nervous system (Söderberg et al. 1990
). For example, in the case of Alzheimer’s disease, a
decrease in DHA in brain phosphatidylethanolamine has been reported
(Söderberg et al. 1991
). The possibility of a
relationship between lipid intake and learning performance in training
tests has also been investigated extensively. In such studies, it was
demonstrated that long-term feeding of fish oil increased and/or
maintained learning ability in mice (Suzuki et al. 1998
). Aged animals, fed an 
-linolenic acid-rich diet, had
an increased learning ability and a longer mean survival time
(Umezawa et al. 1995
, Yamamoto et al. 1991
). These studies suggest that (n-3) polyunsaturated fatty
acids (PUFA) are an essential component for maintaining and improving
brain function in aged animals. However, there have been few
comparative studies of the direct effect of dietary DHA on learning
ability during aging.
The central cholinergic nervous system is known to play a critical role
in cognitive functions such as memory and learning (Drachman and Leavitt 1974
), and impaired cholinergic transmission is thought
to be associated with the memory deficits that occur during aging
(Caird 1966
). Although the rate of acetylcholine
synthesis and release decreases during aging, there are no changes in
its basal concentration (Decker 1987
). The use of
phosphatidylcholine (PC) as a choline source has been proposed because
its consumption does not give subjects a fishy odor (Jope 1982
), and it causes a much greater increase in plasma
choline levels than equivalent doses of choline chloride
(Wurtman et al. 1977
). An increase in avoidance
performance in aged mice fed dietary PC in comparison with control
animals has been reported (Leathwood et al. 1982
).
However, because the PC used in this study originated from soybean,
which also contains -linolenic acid, the effect on learning
performance could be associated with the influence of DHA, which is
synthesized from -linolenic acid in vivo. There is little
information on the direct effect of PC on learning ability in mice of
different ages.
In this study, the effect of diet on learning behavior was studied in animals of different age. The influence of feeding DHA ethyl ester (DHA-EE) and PC from eggs on maze-learning ability in mice was examined, and the additive effect of dietary DHA and PC on the learning task was determined in young and old mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Crj:CD-1 mice aged 3 wk or 14 mo were used. All mice originated
from the same colonies and were obtained from Charles River Japan
(Atsugi, Kanagawa, Japan). Mice (14 mo old) were fed a nonpurified
diet, MF (Oriental Yeast, Tokyo, Japan) in our laboratory for 13 mo.
DHA (DHA-95E, ethyl ester derivative of all
cis-4,7,10,13,16,19-DHA, 95% pure) was obtained from
Harima Chemicals (Tsukuba, Japan). PC (PC-98S, 98% pure) was obtained
from Q. P. (Tokyo, Japan). Twenty-eight 3-wk-old mice were
randomly divided into four groups of seven as follows:
1) a group fed 5 g palm oil/100 g diet (Control
Group); 2) a group fed 2 g DHA-EE/100 g diet plus
3 g palm oil/100 g diet (DHA-EE Group); 3) a group
fed 5 g egg-PC/100 g diet (egg-PC Group); and 4) a
group fed 1 g DHA-EE/100 g diet plus 2.5 g egg-PC/100 g diet
plus 1.5 g palm oil/100 g diet (DHA-EE + egg-PC Group) for 5
mo. Twenty-four 14-mo-old mice were divided into four groups of six
and fed the same four diets as described for the 3-wk-old mice. Each
diet contained 5 g/100 g lipid sources and is presented in Table 1
. The main fatty acid composition of lipids in each diet group is
presented in Table 2
. In the palm oil diet, the main fatty acids were palmitic acid (16:0)
and oleic acid [18:1 (n-9)]. In this study, the control diet was
(n-3) fatty acid deficient, and all diets were deficient in
-linolenic acid. The experimental diets contained different amounts
of DHA (0.9 g/100 g fatty acids in the egg-PC group, 13.8 g/100 g
fatty acids in the DHA-EE + egg-PC group and 23.7 g/100 g fatty
acids for the DHA-EE group).The palm oil, DHA-EE and DHA-EE
+ egg-PC diets contained 5, 3 and 1.5 g/100 g triglyceride,
respectively. The egg-PC diet contained no triglyceride. The
egg-PC and DHA-EE + egg-PC diets contained 5 and 2.5
g/100 g phospholipid, respectively. There was no phospholipid in the
palm oil and DHA-EE diets. For the levels of choline, the palm oil,
DHA-EE, egg-PC and DHA-EE + egg-PC diets contained
0.03, 0.03, 0.71 and 0.37 g/100 g, respectively. The palm oil and
egg-PC diets contained no ethyl ester. The diets were stored at
-25°C and fresh supplies were given to the mice once every 2 d.
The diet and water were given ad libitum. All mice were housed in a
standard environment, in which temperature was maintained at 24 ± 0.5°C, and the relative humidity was kept at 65 ± 5% with 12-h
periods of light and dark. Body weights were measured once a week. All
mice were maintained according to the guidelines for experimental
animals of National Food Research Institute, Japan.
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To determine maze-learning ability in mice, a video tracking and
motion analysis system (EMTEC, Tama, Tokyo, Japan) was used. The
analysis system, measuring the rapid real-time picture acquisition,
was described previously by Suzuki et al. (1998)
. In
this study, we used a new and more complicated maze, which contains
many blind alleys (Fig. 1
). The apparatus consisted of a maze (36 x 50 cm), a shot camera
(Artist G120), image processing equipment (microprocessor for
rapid information processing), a personal computer and a printer. A
program for monitoring the pattern of animal movement and the time
spent getting from the maze entrance to exit was adopted. This program
allowed direct recording of the X-Y coordinates of mouse movement
on a computer disk file. The conditioning of all mice was the training
of mice to drink water and was perfected using a simple maze of three
partition walls 4 mo after the start of the feeding trial. The first
maze trials were done after 24 h of water deprivation so that the
thirsty animals sought water, which was placed outside the maze exit.
The second trial was performed under the same condition on d 4 after
the first trial; the third trial was conducted on d 4 after the second
trial. The time required to reach the exit, the number of times that a
mouse strayed into blind alleys and the behavior of a mouse in the maze
were measured.
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All results were expressed as means ± SD, and
significance was determined by two-way (age x diet) ANOVA
using the SigmaStat statistical program package (Jandel, Erkrath,
Germany). When the F-test was significant, comparisons between control
and each dietary group were done using Dunnett’s test at = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Body weight did not differ among young (44.8 ± 0.4g) or old (47.5 ± 0.5g) mice. The food consumption was 4.3 ± 0.2 g/d in both young and old mice, and the differences among the dietary groups were not significant.
Effect on maze-learning ability in young mice.
During trial 2, the time required to reach the maze exit was
significantly less in the DHA-EE diet group compared with the
control group (P < 0.05) (Fig. 2
). The number of times that a mouse strayed into blind alleys in the
maze was significantly fewer in the DHA-EE or egg-PC groups
than in the control group (P < 0.05) (Fig. 3
). Groups did not differ significantly in time or number in trials 1 and
3 (Figs. 2
and 3)
.
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During trial 2, the time required to find the exit was significantly
less in the DHA-EE, egg-PC and DHA-EE + egg-PC diet groups
compared with the control group (P < 0.05) (Fig. 2)
.
When compared with young mice, the time required to reach the exit was
longer in the palm oil, DHA-EE and egg-PC diet groups of old
mice (P < 0.05), but the time was shorter in the
DHA-EE + egg-PC diet group of old mice (P
< 0.05). In trial 3, the mice fed DHA-EE, egg-PC and
DHA-EE + egg-PC diets took less time to reach the exit
(P < 0.05).
In trial 1, the egg-PC diet group strayed significantly into blind
alleys of the maze fewer times than the control group (P
< 0.05) (Fig. 3)
. During trials 2 and 3, the number of times the
mice strayed into blind alleys was significantly fewer in all
experimental groups compared with the control group (P
< 0.05). In trial 3, the DHA-EE and DHA-EE + egg-PC
groups of old mice strayed into blind alleys fewer times than the young
mice, whereas the palm oil group of old mice strayed into blind alleys
more frequently than young mice (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We assume that
modifications of HUFA in mouse brain after feeding of DHA, particularly
an increase in DHA and a reciprocal decrease in arachidonic acid [20:4
(n-6)] levels, are associated with improved learning ability. Our
previous report suggested that levels of DHA in brain lipids decrease
with increasing age (Suzuki et al. 1989
). Humans with
senile dementia, treated for 6 mo with fish oil capsules (1400 mg
DHA/d) in addition to the usual drugs, showed improvement in
intellectual function (Miyanaga et al. 1995
).
We reported that the majority of DHA incorporated into brain is found
in microsomal, synaptosomal and mitochondrial fractions (Suzuki et al. 1997
) . More recently, we showed that synaptic membrane
fluidity in mice fed a DHA-rich diet was higher than that in
control mice (Suzuki et al. 1998
). This finding implied
that DHA may influence the function of the synaptic membrane, which
plays an important role in maintaining and improving learning ability
and memory. Thus, the superior learning ability of mice fed the
DHA-EE diet observed in this study may be associated with a higher
level of synaptic membrane fluidity.
It has been shown that administration of PC containing 6.1% DHA
improved the short-term memory of mice with dementia, but it did
not alter the memory of normal mice (Chung et al. 1995
).
The improvement in memory associated with the intake of DHA-rich PC
may be due to the influence of DHA on the phospholipids of neuronal
membranes. However, the egg-PC diet used in this study contained
very little (n-3) PUFA, including -linolenic acid and DHA,
suggesting that the increased learning ability of mice fed this PC diet
might be due to a direct effect of PC. A possible explanation for the
enhanced effect of PC on maze-learning ability is that an intake of
PC provides choline, a precursor of the neurotransmitter acetylcholine,
which will therefore be present in increased concentrations in the
brain (Jope 1982
). Thus, it may be that the mechanisms
by which DHA-EE and egg-PC diets improved learning ability are
different.
In old mice, the DHA-EE + egg-PC and egg-PC groups as well as the DHA-EE group took less time to reach the maze exit and strayed fewer times into blind alleys, indicating an increased learning ability. These results suggest that DHA and PC are necessary to maintain and improve learning ability and memory in elderly animals.
In conclusion, this study suggests that the intakes of DHA-EE and egg-PC considerably enhance maze-learning ability and brain functions in old mice.
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FOOTNOTES |
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3 Abbreviations used: DHA, docosahexaenoic acid; DHA-EE, docosahexaenoic acid ethyl ester; HUFA, highly unsaturated fatty acids; PC, phosphatidylcholine; PUFA, polyunsaturated fatty acids.
Manuscript received July 12, 1999. Initial review completed August 17, 1999. Revision accepted December 6, 1999.
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