EPA/DHA and Vitamin A Supplementation Improves Spatial Memory and Alleviates the Age-related Decrease in Hippocampal RXRγ and Kinase Expression in Rats

Abstract

Studies suggest that eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and vitamin A are critical to delay aged-related cognitive decline. These nutrients regulate gene expression in the brain by binding to nuclear receptors such as the retinoid X receptors (RXRs) and the retinoic acid receptors (RARs). Moreover, EPA/DHA and retinoids activate notably kinase signaling pathways such as AKT or MAPK, which includes ERK1/2. This suggests that these nutrients may modulate brain function in a similar way. Therefore, we investigated in middle-aged rats the behavioral and molecular effects of supplementations with EPA/DHA and vitamin A alone or combined. 18-month-old rats exhibited reference and working memory deficits in the Morris water maze, associated with a decrease in serum vitamin A and hippocampal EPA/DHA contents. RARα, RXRβ, and RXRγ mRNA expression and CAMKII, AKT, ERK1/2 expression were decreased in the hippocampus of middle-aged rats. A combined EPA/DHA and vitamin A supplementation had a beneficial additive effect on reference memory but not in working memory in middle-aged rats, associated with an alleviation of the age-related decrease in RXRγ, CAMKII, AKT, and ERK1 expression in the hippocampus. This study provides a new combined nutritional strategy to delay brain aging.

Keywords: hippocampus; kinases; n-3 long-chain PUFA; retinoid receptors; spatial memory; vitamin A.

FIGURE 1

Effect of age and diet (EPA/DHA, Vit A, or EPA/DHA + Vit A) on spatial learning, reference memory and cued learning in the Morris water maze. Figures show (A) the mean distance swum during acquisition to reach a submerged platform located at the same position over 9 days with four trials per day; (B,C) Percentage of time spent in the target quadrant with the platform removed in the probe test over (B) 60 s or (C) 20 s; (D) the mean distance swum during the cued learning. Values are mean ± SEM, n = 8–10 rats per group. Data were analyzed by (A) repeated measures ANOVA or (B–D) one-way ANOVA followed by the Fischer PLSD post hoc test. Signs indicate values different from (A) day 1 or (B,C) from Adult control: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 and from middle-aged EPA/DHA: §p < 0.05; or compared with chance level by a Student’s one group t-test: #p < 0.05; ##p < 0.01; ###p < 0.001.

FIGURE 2

Effect of age and diet (EPA/DHA, Vit A, or EPA/DHA + Vit A) on spatial working memory (matching-to-place version) in the Morris water maze. Figures show (A) the mean distance swum to reach a submerged platform over 3 days in the first trial (T1) followed by a second trial (T2) with an inter-trial interval (ITI) of 30 s; (B) the mean distance swum to reach a submerged platform over 3 days in the T1 and T2 with an ITI of 2 min. The platform was moved to a different location each day. Values are mean ± SEM, n = 8–10 rats per group. Data were analyzed by Student’s paired t-test: different from T1: ^∗p < 0.05; ^∗∗p < 0.01.

FIGURE 3

Effect of age and diet (EPA/DHA, Vit A, or EPA/DHA + Vit A) on serum retinol concentrations. Values are mean ± SEM, n = 8–10 rats per group. Data were analyzed by a one-way ANOVA followed by the Fischer PLSD post hoc test. Signs indicate values different from Adult control: ^∗∗∗p < 0.001 and from middle-aged control: #p < 0.05.

FIGURE 4

Effect of age and diet (EPA/DHA, Vit A, or EPA/DHA + Vit A) on the mRNA expression of RARα, β, γ (A), and RXRα, β, γ (B). The mRNA levels are expressed as the percentage of Adult control mRNA expression of target/reference ratio normalized by the calibrator. Values are mean ± SEM, n = 8–10 rats per group. Data were analyzed by one-way ANOVAs followed by the Fischer PLSD post hoc test. Signs indicate values different from Adult control: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 and from middle-aged Vit A and middle-aged EPA/DHA: §p < 0.05.

FIGURE 5

Effect of age and diet (EPA/DHA, Vit A, or EPA/DHA + Vit A) on the mRNA expression of ERK1, ERK2 (A) and CAMKII and AKT (B). The mRNA levels are expressed as the percentage of Adult control mRNA expression of target/reference ratio normalized by the calibrator. Values are mean ± SEM, n = 8–10 rats per group. Data were analyzed by one-way ANOVAs followed by the Fischer PLSD post hoc test. Signs indicate values different from Adult control: ^∗p < 0.05; ^∗∗p < 0.01, from middle-aged control: ##p < 0.01 and from middle-aged EPA/DHA: §p < 0.05; §§p < 0.01.

FIGURE 6

Effect of age and diet (EPA/DHA, Vit A, or EPA/DHA + Vit A) on protein kinase expression in the hippocampus analyzed by Western blot. The upper panel (A) illustrates representative immunoblots probed for phospho-Erk1/2, Erk1/2 and actin. Hippocampal Erk1/2 (B), phospho-Erk1/2 (C), CAMKII (D), phospho-CAMKII (E), AKT (F), and phospho-AKT (G) protein levels were normalized against actin and expressed as a percentage of adult control expression of the same protein. Values are means ± SEM, n = 6–9 rats per group. Data were analyzed by one-way ANOVAs followed by the Fischer PLSD post hoc test. Signs indicate values different from Adult control: ^∗p < 0.05; ^∗∗p < 0.01; from middle-aged control: ##p < 0.01 and from middle-aged EPA/DHA: §p < 0.05; §§p < 0.01.