MR 7179 Centre National de la Recherche Scientifique, Muséum National d’Histoire Naturelle, Brunoy, France
i g h l i g h t s
Fish oil supplementation acted on exploration when started late in life of primates.Fish oil supplementation did not improve anxiety and memory in old primates.Fish oil supplementation affected exploration activity differently across aging.
r t i c l e i n f o
rticle history:eceived 8 June 2012eceived in revised form 4 August 2012ccepted 10 August 2012vailable online 17 August 2012
The data are inconsistent about the ability of dietary omega-3 fatty acids to prevent age-associated cog-nitive decline. Indeed, most clinical trials have failed to demonstrate a protective effect of omega-3 fattyacids against cognitive decline, and methodological issues are still under debate. In contrast to humanstudies, experiments performed in adult rodents clearly indicate that omega-3 fatty acids supplement canimprove behavioural and cognitive functions. The inconsistent observations between human and rodentstudies highlight the importance of the use of non-human primate models. The aim of the present studywas to address the impact of omega-3 fatty acids (given in the form of dietary fish oil) on exploratoryactivity, emotional status and spatial reference memory in the aged mouse lemur, a non-human primate.
Aged animals fed fish oil exhibited decreased exploratory activity, as manifested by an increase in thelatency to move and a reduced distance travelled in an open-field. The fish oil-supplemented animalsexhibited no change in the anxiety level, but they were more reactive to go into the dark arms of a
light/dark plus-maze. In addition, we found that fish oil supplementation did not significantly improvethe spatial memory performance in the Barnes maze task.
This study demonstrated for the first time that a fish oil diet initiated late in life specifically modifies theexploratory behaviour without improving the spatial memory of aged non-human primates. Omega-3fatty acid supplementation may be effective when started early in life but less effective when started atlater ages.
. Introduction
The omega-3 polyunsaturated fatty acids (n-3 PUFA) are impor-ant nutrients that ensure the optimal maturation of brain functionsnd may have a beneficial effect on the incidence of neuropsychi-tric and neurodegenerative disorders [1]. In vertebrates, includingrimates, the neuron and astrocyte membranes contain high con-
entrations of long-chain PUFA of the n-3 and n-6 series, mainlyocosahexaenoic acid (DHA, 22:6 n-3) and arachidonic acid (20:4-6) [2,3]. The specific accretion of DHA (the main n-3 PUFA in brain
∗ Corresponding author at: UMR 7179 Centre National de la Recherche Scien-ifique, Muséum National d’Histoire Naturelle, 1 avenue du Petit Château, 91800runoy, France. Tel.: +33 0160479248; fax: +33 0160479218.
neuronal, glial and vascular membranes) during perinatal devel-opment is considered essential for the proper functioning of themammalian central nervous system and may thus be of criticalimportance in the ageing brain. In rodents, a dietary deficiency inn-3 PUFA leads to lower levels of brain membrane DHA, which isaccompanied by behavioural alterations, such as poor performancein learning tasks and brightness discrimination as well as changes inthe physiological properties of receptors, transporters and enzymesin neuronal membranes (reviewed in [1]). The importance of n-3PUFA in the ageing brain is revealed by epidemiological studies thatdemonstrate the protective effects of fish and DHA against the riskof Alzheimer’s disease [4–6]. However, data are divergent about the
ability of dietary n-3 fatty acids to prevent age-associated cognitivedecline. Indeed, most clinical trials have failed to demonstrate aprotective effect of n-3 PUFA dietary supplements against cognitivedecline, and methodological issues are still under debate [5,7].
In addition to human studies, experimental studies in adultodents have generally demonstrated beneficial effects of n-3 fattycid diets (e.g., fish oil) on behaviour, including locomotor andxploratory activity, anxiety and cognitive functions. Indeed, proto-ols using n-3 fatty acid-deficient diets show that deficient animalsxperience higher anxiety levels and more cognitive deficits thanontrol animals (reviewed in [8]). However, the influence of n-
fatty acid-enriched diets on behaviour, and particularly onognitive functions, is not as clear. In adult rats that were previ-usly exposed to a deficient diet, n-3 fatty acid supplementationmproved spatial reference memory in the Morris water maze test9,10] and the radial arm maze [11,12]. However, additional stud-es in adult rodents have suggested that n-3 supplementation didot provide any benefit to associative learning [13] or spatial refer-nce learning and memory tasks using the Morris water maze andircular platform tests [13–16].
To the best of our knowledge, only two studies have shown positive effect of fish oil or n-3 fatty acids (i.e., DHA, and EPA:icosapentaenoic acid) on the cognitive abilities of aged animals.upplementation with DHA for 7 weeks reduced the number ofrrors in a passageway water maze test in 15-month-old mice [17].ikewise, Gamoh et al. showed that 25-month-old aged rats pro-ided with a diet supplemented with DHA for 5 weeks expressedess reference and working memory errors in the radial arm maze.owever, this improvement was only observed in the later phasef learning, after more than 21 days of training with two daily tri-ls [18]. These studies suggest that n-3 fatty acid diets reduce theevelopment of cognitive deficits, even when the administrationegan in old age. In contrast to these experiments, other studiessing fish oil supplementation did not detect an improvement inpatial learning or memory in the Morris water maze test in 22-onth-old mice [19], 24-month-old rats [9] or 28-month-old rats
20]. For example, a fish oil diet for 1 month did not improve spatialearning and memory in the Morris water maze in 24-month-oldats, whereas 2-month-old rats given the fish oil diet performedetter than control animals [9].
The inconsistencies in the results between the human andodents studies (in both adult and aged subjects) emphasize themportance of the use of non-human primate models. To date, veryew studies have investigated the impact of n-3 PUFA supplemen-ation on the behavioural and cognitive functions of non-humanrimate species. Among them, Tsukada et al. demonstrated thatupplementing aged monkeys with DHA for 1–4 weeks (short-termietary supplementation) led to increased regional cerebral bloodow, a parameter closely linked to neuronal activation [21]. Fur-hermore, we recently demonstrated that 6 months of n-3 PUFAietary supplementation significantly reduced signs of anxiety and
mproved performance in spatial memory tasks in a non-humanrimate model, the grey mouse lemur [22]. However, it is notnown whether the n-3 PUFA diet can effectively counteract age-elated decline in aged non-human primates.
The grey mouse lemur is a nocturnal prosimian primateriginating from Madagascar. This non-human primate has anpproximate life expectancy of 8 years and is characterised by itsmnivorous dietary habits and small size and weight (80–120 g).his species is commonly used as a model of cerebral ageing22–25]. During ageing, some mouse lemurs develop spontaneousrain and cognitive impairments similar to those observed in neu-odegenerative diseases in humans. Massive cerebral atrophy, aign of a neurodegenerative process, can be detected in some agednimals [23]. This atrophy is predictive of behavioural and cogni-ive impairments in aged animals [24]. These cognitive disorders
ave been extensively reviewed elsewhere [25]. It has been shownhat mouse lemurs mimic the pattern of cognitive ageing describedn humans, namely intact procedural memory, progressive and
idespread decline in executive functions, and limited declarative
esearch 235 (2012) 280– 286 281
memory dysfunction, except in case of age-associated pathologiessuch as Alzheimer disease. It is noteworthy that not all the agedmouse lemurs are equally affected by behavioural and cognitiveageing (similar to other species, including humans).
The aim of the present study was to address the impact of n-3fatty acids (given in the form of dietary fish oil) on the exploratoryactivity, emotional status and spatial reference memory in agedmouse lemurs. In parallel, the plasma fatty acid status of the animalswas evaluated. To the best of our knowledge, this study is the firstto explore the impact of n-3 fatty acid supplementation on anxietyand spatial memory in an aged non-human primate.
2. Materials and methods
2.1. Animals and experimental design
Twelve aged female grey mouse lemurs (Microcebus murinus) born and reared inthe Brunoy colony (MNHN, France, license approval N◦ A91.114.1) were used in thisstudy. The aged category was based on the survival data measured in the breedingcolony of Brunoy [25]. An analysis of the survival of 361 female mouse lemurs indi-cated that the median survival time is 4.9 years, and the maximum lifespan recordedwas 12 years. Animals were raised on fresh fruits and a laboratory daily-made mix-ture of cereals, milk and egg. All experiments were carried out in accordance withthe European Communities Council Directive (86/609/EEC) and were performedunder authorisation N◦ 91–582 delivered by the “Direction Départementale de laProtection des Populations de l’Essonne”.
Two weeks before the experiments began, the animals were provided daily withfresh fruit (6 g of apple and 6 g of banana) and 15 g of a mixture composed of cereals,milk and egg. Water was provided ad libitum. The animals were housed in individualcages (50 × 49 × 50 cm) with wooden branches and wooden nests at standard con-ditions of temperature (24–26 ◦C) and relative humidity (55%). The animals wereweighed at the beginning of the experiment and every 2 weeks throughout theexperiment (without interfering with the behavioural tests).
After the first session of behavioural characterisation (week 0), the animals weredivided into two homogeneous groups of six animals each: the control and fishoil-diet groups. The mouse lemurs were randomly assigned to the two experimen-tal groups according to their age (mean = 6.7 ± 0.6 and 6.5 ± 0.7 years old; range:5–9.4 and 4.9–8.9 years old, respectively for the control and fish oil diet groups) andweight (mean = 93.8 ± 6.8 and 102.0 ± 7.5 g, respectively for the control and fish oildiet groups). Moreover, no behavioural differences were observed between the twogroups in the parameters of each task during the first behavioural session, assuringhomogeneous groups. Examinations of the eyes of the twelve mouse lemurs by aveterinary ophthalmologist did not detect any anomalies that would affect visualacuity.
Before the supplementation (first session, week 0) and after 14 weeks of sup-plementation (second session, week 15), the animals were tested in each of thethree tasks described below. All behavioural tests were carried out during the last4 h before the daily active phase, with an interval of 24 h between two consecu-tive tests, and in the following chronological order: open-field test, Barnes mazetest and light/dark plus-maze test. All of the experiments were performed by anobserver blind to the diets, and the animals were evaluated in a random order eachday. Averaging all of the weekly weight determinations, there were no overall dif-ferences between the groups, and there were no differences in the weights at theend of the study (data not shown). Behavioural and cognitive tests and data analysishave been performed by experimenter that was blind to the dietary supplement.
2.2. Dietary protocol
The fish oil-supplemented group then received the laboratory-made food sup-plemented with fish oil (OMEGAVIE tuna oil, Polaris, Pleuven, France), which is richin long-chain n-3 PUFA, while the control group received food supplemented forisoenergy with the same volume of olive oil (rich in monounsaturated fatty acidsand poor in n-3 fatty acids). In the fish oil-supplemented group, the intakes ofeicosapentaenoic acid (EPA, 20:5 n-3) and of docosahexaenoic acid (DHA, 22:6 n-3)represented approximately 0.06% and 0.3% of the total energy, respectively, whichis equivalent to the highest level of consumption of French coastal populations [26]and corresponds to the recommended daily intake for the French population [27].These proportions correspond to a daily intake of 6 mg EPA and 30 mg DHA peranimal.
2.3. Lipid analysis
After the last behavioural tests, the blood was collected in heparinized tubes and
centrifuged, and the plasma was stored at −80 ◦C until analysis. The total lipids wereextracted from the plasma with chloroform/methanol 2/1 using the Folch method.The total plasma phospholipids were isolated by solid phase liquid chromatographyon silica cartridges; sequential elution was performed with chloroform and thenwith methanol, which contained the phospholipid fraction [28]. All eluents were
2 Brain Research 235 (2012) 280– 286
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Table 1Fatty acid composition (g/100 g) of the plasma phospholipids (PL).
Fatty acids representing less than 0.5 g/100 g were not reported; SFA, saturated
82 S. Languille et al. / Behavioural
ried under nitrogen, and the phospholipid fractions were transmethylated with0% boron trifluoride (Fluka, Sokolab) at 90 ◦C for 20 min [29]. Fatty acid methylsters were analysed by gas chromatography [30]; the fatty acid compositions arexpressed as the weight percentage (g/100 g total fatty acids).
.4. Open-field test
The Plexiglas® apparatus had a beige floor of 95 × 95 cm. The 25-cm high wallsere also painted beige and covered with a transparent ceiling. Four 60 W white
ights were placed in each corner of the system, allowing homogeneous illuminationf the open field. Before each test, the whole area was cleaned.
Each mouse lemur was individually placed in a corner of the open-field via anirlock (112 cm2). After a 30-s resting phase, the door was removed. The latency ofhe first movement (distance of at least 14 cm) and the distance travelled during the-min session were recorded using a video camera and Ethovision XT7.
.5. Light/dark plus-maze test
The apparatus was made of plastic with four cylindrical arms that were 30-cmong and 9 cm in diameter; thus, the animals could not escape from the apparatus.he arms were arranged in a cross-like formation, with two dark opposite armsblack walls) and two light arms (transparent walls) that intersected at a centralquare platform (16.5 × 16.5 cm), which provided access to any of the four arms.ll of the floor surfaces were black, and the central platform was under homo-eneous illumination at 60 W. The maze was enclosed in a four-walled chamber66 × 66 × 41 cm) to prevent use of extra-maze cues for exploration. To avoid theresence of different odour stimuli in the two types of arms, the four arms were firstaturated with a mixture of urine from other mouse lemurs. Moreover, between eachest, the maze was cleaned, and the position of the types of arms was randomised.
Each mouse lemur was individually placed in the central platform via an airlock143 cm2). After 30 s, the doors were slowly raised, and the mouse lemur was allowedo freely explore the plus-maze for 5 min while its behaviour was video-recorded.he variables recorded were the following: the latency before the first visit to theark arms and light arms and the number of entries and time spent (with all fouraws) inside each arm. If the animal did not visit the arm, a latency of 300 s was
ndicated. The percentages of time spent in the light arms and light-arm entriesere each calculated in relation to the total time spent in both types of arms and
he number of entries into both types of arms.
.6. Barnes maze test
The apparatus was adapted for mouse lemurs [24] from the device described byarnes. It consisted of a white circular platform (Ø 100 cm) containing 12 equallypaced circular holes (Ø 4.5 cm) on the periphery of the platform. Black Plexiglasestboxes (10.5 × 10.5 × 20 cm), which served as refuges, were inserted beneathach hole to avoid the use of olfactory perception to find the target. The platformas divided into 12 compartments (1 hole/compartment) with transparent Plexi-
las walls (25 cm high × 24.5 cm long) to prevent the use of the “clock” navigationtrategy (walking along the periphery and inspecting each hole one by one). To pre-ent the mouse lemurs from escaping, the platform was entirely surrounded by a5-cm high white wall across its circumference and covered with a transparent Plex-
glas ceiling. The platform was enclosed in a black chamber (122 × 122 cm) with ane-way mirror window to allow observation. Thirty-two evenly spaced 2 W lightsere affixed beneath the one-way mirror and following the circular perimeter of
he maze (50 cm above the platform) to illuminate the maze. Moreover, the centref the maze was also illuminated with a 60 W light. Forty objects (different forms,izes and colours) were attached to the black chamber wall around the periphery ofhe platform (50 cm above) to serve as extra-maze visual cues. Before each trial, thelatform and the target box were cleaned, and the platform was randomly rotatedn its central axis to avoid the use of intra-maze cues.
In each trial, the mouse lemur was individually placed in the centre of the plat-orm via an airlock (83 cm2). After 30 s, the airlock was lifted to release the animal.f the animal did not explore the maze after 2 min, the maze was gently shaken. Thenimal had to reach the target box positioned beneath one of the 12 holes, whichas kept constant in relation to the visual cues in all trials. When the animal entered
he target compartment, the target box was opened to allow the animal to escaperom the maze and the trial was stopped; the animal remained in the target box for
min.On the first day, each animal was given two 5-min trials of learning in a four-
alled chamber containing only the open target compartment (one-choice test),hich provided access to the target box. The short-term (3 min after the last learning
rial) and long-term (24 h later) memories were evaluated by a 10 min trial involving2 open compartments, but with only the target compartment providing accesso the target box. The latency to reach the target compartment was determinedmaximum of 600 s), as was the total number of errors (e.g., the number of entriesnto compartments containing the non-escape holes).
fatty acids; MUFA, Monounsaturated fatty acids; PUFA, Polyunsaturated fatty acids.Data are presented as the means ± SEM (n = 6). * represents a statistically significantdifference between the dietary treatments (p < 0.05).
2.7. Statistical analysis
For all statistical assessments, data were first assessed for normality using R2.12.1 software. Unpaired T-tests were performed to compare the plasma fattyacid content in the control and fish oil-supplemented animals. Since behaviouraldata were not normally distributed, we performed non-parametric tests. TheMann–Whitney analysis (W-value) was performed to compare the data betweenthe control and the fish oil-diet groups. The time effect was evaluated by the pairedWilcoxon signed rank test (V-value), as a post hoc test of the Friedman test. TheFisher’s exact test was used to differentiate the proportion of animals which madesuccessful trials. A P-value of < 0.05 was considered significant. Plasma lipids wereexpressed as mean and SEM. All values of the behavioural tests are given as themedian and interquartile (IQ: lower quartile-upper quartile) in the text and arerepresented by box plots in the figures.
3. Results
3.1. Plasma lipids
The quantification of the fatty acids in the total plasma phos-pholipids (Table 1) demonstrated that fish oil supplementationsignificantly increased the level of n-3 fatty acids and compen-satorily decreased levels of n-6 and monounsaturated fatty acids.The n-3 fatty acids level was 3.7-fold higher in the fish oil-supplemented animals than in the controls (23.7 ± 1.3% of the totalfatty acids in the supplemented animals versus 6.4 ± 0.8% in thecontrols, p = 0.007), while the total n-6 levels were lower in the fishoil diet group than in the control group (p = 0.014), and the levelsof monounsaturated fatty acids were not different. More specif-ically, DHA represented 3.9 ± 0.6% of the total fatty acids in thecontrols and 17.2 ± 1.4% in the supplemented animals (p = 0.014),while arachidonic acid represented 24.5 ± 1.5% of the total fattyacids in the controls and 16.2 ± 2.6% in the supplemented animals(p = 0.03). The ratio of the total n-6: total n-3 PUFA was equal to1.3:1 in the fish oil-supplemented animals and 7:1 in the controlgroup (p = 0.015). The saturated fatty acids were not significantlyaltered by the dietary treatment.
3.2. Open-field test
The distance travelled did not change across the sessions (week0 vs. week 15; V = 8, p = 0.18), but the latency of the first movement
S. Languille et al. / Behavioural Brain Research 235 (2012) 280– 286 283
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Fig. 1. Effect of a fish oil diet on exploratory activity in the open-field test in agedmouse lemurs. (A) Latency (s) of the first movement. (B) Distance travelled (cm).Control (n = 6) and fish oil diet (n = 6) animals were tested over a 5-min session, atweek 0 (baseline) and week 15 (after 14 weeks of fish oil supplementation). Sig-nificant differences between the groups (Mann–Whitney analyses) are indicatedai
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Fig. 2. Effect of the fish oil diet on anxiety-related behaviour in the light/dark plus-maze test in aged mouse lemurs. The latencies (s) of the first entry into the dark arm(A) and light arm (B) were evaluated in the control (n = 6) and fish oil (n = 6) animalsat week 0 (baseline) and week 15 (after 14 weeks of fish oil supplementation). Sig-
257.5 s, IQ: 73.3–300 s in the control group; median: 209.5 s, IQ:
s * (p < 0.05). Significant differences between the sessions (Wilcoxon analysis) arendicated as § (p < 0.05).
ncreased significantly across the sessions, from a median of 55 sIQ: 15.8–300) in week 0 to a median of 300 s (IQ: 168–300 s; V = 26,
= 0.0499; Fig. 1A) in week 15, suggesting that the behaviour ofll of the animals evolved independently of the dietary treatmentsetween the two open-field sessions.
Before supplementation (week 0), both groups expressed no dif-erences for the latency of first movement (p = 0.25; Fig. 1A) and forhe distance travelled (p = 0.94; Fig. 1B).
After 14 weeks of supplementation (week 15), the latency ofhe first movement was significantly higher in the fish oil dietroup (median: 300 s, IQ: 300–300 s) compared to the control groupmedian: 149 s, IQ: 43–271.5 s; W = 29; p = 0.0495; Fig. 1A). More-ver, the fish oil-supplemented animals exhibited a tendency toxplore less during the 5 min session. The median distance travelledas 17.5 cm (IQ: 3.8–230 cm) in the control and 0 cm (IQ: 0–0 cm)
n the fish oil-supplemented group (W = 8.5; p = 0.09; not signifi-antly different; Fig. 1B), and the median duration of movementas 1.7 s (IQ: 0.2–4.2 s) in the controls and 0 s (IQ: 0–0 s) in the
nificant differences between the groups (Mann–Whitney analyses) are indicated as* (p < 0.05).
supplemented group (W = 8; p = 0.07; not significantly different;data not shown).
3.3. Light/dark plus-maze test
No differences were found between the first (before sup-plementation, week 0) and second sessions (after 14 weeks ofsupplementation, week 15) in this task regardless of the parameterconsidered (p > 0.5).
Before supplementation (week 0), both groups expressed no dif-ferences for the latency of the first entry into the dark arm (p = 0.87;Fig. 2A) and for the latency of the first entry into the light arm(p = 0.23; Fig. 2B).
In the week 15 condition, there was a significant group effect onthe latency of the first entry into the dark arm, with the fish oil sup-plementation decreasing the latency (median: 167 s, IQ: 13.8–300 sin the control group; median: 2 s, IQ: 0–12.3 s in the supplementedgroup; W = 4; p = 0.02; Fig. 2A). In contrast, there were no significantdifferences in the latency of the first entry into light arm (median:
114.5–300 s in the supplemented group; W = 19.5; p > 0.5; Fig. 2.B).The Chi-square test revealed that most of the animals spent signif-icantly more time in the dark arms than in the light arms (83% of
284 S. Languille et al. / Behavioural Brain Research 235 (2012) 280– 286
Table 2Effect of the fish oil diet on success in the Barnes maze test in aged mouse lemurs.
Number ofsuccessfulanimals/total
Control Fish oil Fisher’stest
Week 0 3 min 6/6 4/6 p = 0.4524 h 5/6 5/6 p = 1
Week 15 3 min 4/6 5/6 p = 124 h 2/6 6/6 p = 0.06
Numbers of control and fish oil-supplemented animals that found the target duringthe 10-min test, after a 3-min or 24-h delay, at week 0 (baseline) and week 15 (after1f
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Fig. 3. Effect of fish oil diet on short- and long-term spatial memories in the Barnesmaze test in aged mouse lemurs. (A) The latency (s) to reach the target. (B) Number
lemurs, we reported that fish oil supplementation did not modify
4 weeks of fish oil supplementation). P-values from the Fisher’s tests are indicatedor the group comparisons.
he fish oil diet animals and 50% of the control animals), but theontrol and fish oil diet groups did not differ in the percentage ofime spent in the light arms or in the percentage of light arm entriesp > 0.5; data not shown).
.4. Barnes maze test
No differences in any of the analysed parameters were observedetween the two sessions in the Barnes maze test (week 0 vs. week5, p > 0.5).
Animals who found the target during the 10 min session wereonsidered successful (Table 2). After supplementation, no differ-nces between the control and fish oil diet animals were observedn the success rate after a 3 min delay, but the control group tendedo find the target compartment less frequently than fish oil dietnimals when tested after a 24 h delay (2/6 animals found the tar-et compartment in the control group vs. 6/6 in the supplementedroup, p = 0.06).
In the week 15 condition, the latency to reach the target andhe number of errors in the control animals did not differ betweenhe tests after 3-min or 24-h delays (V = 2, p = 0.4 for latency,ig 3 A; V = 6.5, p = 0.5 for errors, Fig. 3.B). Similarly, the fish oil-upplemented animals did not alter their behaviour between the-min test and the 24-h test (V = 15, p = 0.4 for latency and errors,ig. 3A and B).
Regardless of the retention delay, no significant differences wereound between the control and supplemented groups. After a 3-
in delay, the control animals reached the target in 200.5 s (IQ:42.75–502.75 s) and made 4 errors (IQ: 0.75–7.25 errors) beforending the target, and the fish oil-supplemented animals reachedhe target in 240.5 s (IQ: 163.5–301.75 s) and made 4.5 errors (IQ:.75–5 errors; W = 18, p = 1 for latency and errors; Fig. 3A and B).fter a 24-h delay, the control animals found the target in 600 s (IQ:08.5–600 s) after 5.5 errors (IQ: 3.25–7 errors), whereas the fishil animals found the target in 209.5 s (IQ: 170.75–234.75 s) after
errors (IQ: 1.25–3.5 errors; W = 10.5, p = 0.2 for latency, Fig. 3A; = 11, p = 0.3 for errors, Fig. 3B).
. Discussion
The present study aimed to determine the effects of dietary fishil supplementation on the exploratory activity, emotional statusnd spatial memory of aged mouse lemurs. Our results revealedhat fish oil supplementation slightly decreased the exploratoryehaviour in a novel environment, as revealed by the significantlyigher latency before moving and the trend to a decreased durationf movement in an open-field. The supplemented animals exhib-ted no change in the anxiety levels, but they were more reactive to
o into the dark arms in the light/dark plus-maze test. In addition,e observed that the fish oil supplementation did not significantly
mprove the spatial memory performance in the Barnes maze task.
of errors prior to reaching the target. Control (n = 6) and fish oil (n = 6) animals weretested 3 min and 24 h after training for the spatial location of the target at week 0(baseline) and week 15 (after 14 weeks of fish oil supplementation).
The plasma fatty acid determination showed that the fishoil-supplemented animals exhibited significantly higher levels ofplasma long chain n-3 PUFA (including EPA, 22:5n-3 and DHA)compared to controls. It has been demonstrated in several mammalspecies that an increase in dietary DHA correlates with the plasmaDHA content, which is in turn predictive of the internal organ DHAstatus and is also a useful biomarker for the brain DHA status[31]. The increased levels of n-3 PUFA in the fish oil-supplementedanimals occurred at the expense of both n-6 PUFA and monoun-saturated fatty acids. These changes contributed to improving thebalance between n-3 and n-6 PUFA in the plasma of the fish oil-supplemented animals to a ratio of n-6: n-3 of 1.3:1 compared to7:1 in the control group. The recommended dietary ratio of n-6: n-3fatty acids should be close to 1:1 for human health benefits [32].
We observed that fish oil supplementation tended to increasethe success (ability to find the target during the 10-min session) inthe Barnes maze test in aged mouse lemurs without improving thespatial memory performance. This observation is consistent withtwo clinical trials in cognitively healthy elderly subjects [33,34].The two human studies did not find a positive effect of n-3 fattyacid supplementation for 6.5 months [33] and 24 months [34] onany cognitive aspects (attention, memory or executive function) insubjects aged 65 years or older. In a previous study in adult mouse
the latency to reach the target or the number of errors in the Barnesmaze task but significantly improved the frequency of successfultrials [32], which is consistent with the present results. Our results
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ndicate that fish oil supplementation in adult and in aged mouseemurs had no effect on memory performance in a low-stress taskut improved the rate of success, which can be a sign of decreasednxiety or increased arousal and/or motivation.
The open-field and plus-maze tests are widely used to assessxploratory and anxiety-related behaviours in rodents. Very fewtudies have investigated the impact of n-3 fatty acid supplemen-ation on these behaviours in aged animals. Carrié et al. showed thatsh oil supplementation beginning in the perinatal period tendedo decrease the exploratory activity in the 5-min open-field testnd significantly decreased the locomotor activity in a 20-min cageest in 17- to 19-month-old mice [35]. Conversely, in 1-month-old
ice, the amount of locomotor activity was significantly highern the fish oil-fed animals than in the control group. This studyuggests that the fish oil diet affects behaviour differently acrossgeing, increasing locomotor activity in adults and decreasing thisctivity in aged mice [35]. Consistent with the adult rodent stud-es, our previous experiments in adult mouse lemurs showed that
hen initiated in adulthood, a fish oil diet increased the explorationn the open-field test [22]. In the present study, the latency beforehe first movement in the open-field test was significantly higher inhe fish oil-supplemented animals compared to the control group.
oreover, these animals exhibited a tendency to travel less dur-ng the 5-min period compared to the controls. This observation ofeduced exploratory activity in aged animals fed with the fish oiliet at a late age supports the Carrié’s study in rodents and stronglyuggests that the impact of n-3 fatty acids supplementation differsccording to age.
Exploratory activity is dependent on arousal, curiosity and stressevel. In adults, the effects of n-3 fatty acids diet on anxiety param-ters in the elevated plus-maze test remain contradictory. Fish oilupplementation can counteract the anxiogenic effects of an n-
fatty acid-deficient diet [13,36] or of a restraint stress protocol10]. In contrast, other studies have shown that n-3 fatty acid-nriched diets did not affect anxiety-related behaviour [15,37–40].ur study is the first to test the impact of n-3 fatty acid supple-entation on anxiety in aged animals. We did not observe any
hanges in the percentage of time or entries into the light arms inhe light/dark plus-maze test, two anxiety parameters, suggestingo anxiolytic effect of the fish oil diet in aged animals. Our findingorroborates the single clinical investigation in older individuals,hich showed no effect of 6.5 months of n-3 fatty acids supple-entation on depression or anxiety, as assessed by questionnaires,
n subjects aged 65 years or older [41]. Thus, it appears that thehanges in the exploratory behaviour of aged animals fed with thesh oil diet could not be explained by a modification of the stress
evel.Paradoxically to the decrease in the exploratory behaviour in
he open-field test, the mouse lemurs fed the fish oil diet took lessime to go into the dark arms of the light/dark plus-maze than theontrol animals. Our results showed that the behaviour of the agednimals might vary with the task and possibly based on previousxposure to each task. To be sure that both group were homoge-eous, we performed an initial behavioural session prior the fish oilupplementation. No differences across the sessions were observedn the plus-maze or Barnes maze, but we observed a decrease inhe locomotor activity in the open-field test between the first andecond session. This change led to the idea that mouse lemursemember the apparatus and adapt their behaviour 15 weeks later.n this context, the fish oil-supplemented animals could exhibit
ore appropriate behaviour as a function of the environment. Athe second session, the fish oil animals may have been less moti-
ated to explore the open field and more alert to enter the preferredrms (dark arms). Likewise, the increase in successful trials in thearnes maze in the supplemented mouse lemurs may have beenue increased arousal or enhance motivation to find the refuge,
esearch 235 (2012) 280– 286 285
suggesting that the fish oil diet may play a role in arousal and/ormotivation status.
Based on recently published data in mouse lemurs placed understandard feeding conditions (control diet), it is likely that the neu-ral tissue requirements for DHA are almost completely met by thecontrol diet [3]. If this speculation were correct, supplementing thediet with fish oil would result in only a modest gain in DHA inthe neural tissues, which might explain the modest gains in cog-nitive performance and behaviour observed in the present study.Several molecular and cellular mechanisms by which n-3 PUFA canhave an impact on neuronal functions have been described suchas the modulation of membrane biophysical properties, regulationof neurotransmitter release, synthesis of biologically active oxy-genated derivatives, and nuclear receptor-mediated transcriptionof genes responsive to fatty acids or to their derivatives (reviewedin [1]), The amplitude of these different mechanisms seems to bemodulated by age since aged animals have a lower response tothe dietary supplementation compared to younger one [22]. It hasbeen recently demonstrated in humans, that DHA supplied by foodis retained longer in the blood and undergoes more �-oxidationand more apparent retroconversion than in young adults [42], whatmay explain age-related differences observed in our studies.
5. Conclusions
This study demonstrates for the first time that a fish oil diet ini-tiated late in life specifically modifies the exploratory behaviourwithout improving the spatial memory performance of aged non-human primates. N-3 fatty acid supplementation may be effectivewhen initiated early in life (from the perinatal period) but less effec-tive when started at a later age. Our recent findings in young adultgrey mouse lemurs corroborate this hypothesis since we recentlydemonstrated that n-3 PUFA dietary supplementation significantlyreduced signs of anxiety and improved performance in spatialmemory tasks in this species [22]. Structural and functional modifi-cations of the brain (e.g., neurotransmission activity [43,44]) duringageing could explain why fatty acids differentially affect behaviouras a function of age. Aged animals may differ from young animalsbecause of ageing-related changes in n-3 fatty acid metabolism. Dif-ferences in plasma incorporation of long-chain n-3 PUFA betweenyoung and aged subjects has been recently described [45], withhigher levels observed in aged healthy subjects. However, to thebest of our knowledge, differences in the brain incorporation of n-3 PUFA between young and aged individuals (in either animal orhuman studies) have never been described.
Acknowledgements
This work was financially supported by the Groupe Lipides etNutrition (part of the Association Franc aise pour l’Etude des CorpsGras) of the Centre National de la Recherche Scientifique/MuséumNational d’Histoire Naturelle.
We thank Sabine Chahory, from the National Veterinary Schoolof Alfort, for performing the eye examinations and Jean-LucPicq, Eric Gueton, Léonie Lelli and Julia Marchal for their helpwith the apparatus construction. We also would like to spe-cially thank Sheherazade Benatia and Jean-Charles Martin, fromthe laboratory «Nutriments Lipidiques et Prévention des MaladiesMétaboliques», plateau BioMeT (BIOlogie Métabolique Timone),UMR INRA 1260/INSERM 1025/Universités Aix-Marseille I & II, forperforming the fatty acid analyses.
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