Dietary levels of pure flavonoids improve spatial memory performance and increase hippocampal brain-derived neurotrophic factor

Abstract

Evidence suggests that flavonoid-rich foods are capable of inducing improvements in memory and cognition in animals and humans. However, there is a lack of clarity concerning whether flavonoids are the causal agents in inducing such behavioral responses. Here we show that supplementation with pure anthocyanins or pure flavanols for 6 weeks, at levels similar to that found in blueberry (2% w/w), results in an enhancement of spatial memory in 18 month old rats. Pure flavanols and pure anthocyanins were observed to induce significant improvements in spatial working memory (p = 0.002 and p = 0.006 respectively), to a similar extent to that following blueberry supplementation (p = 0.002). These behavioral changes were paralleled by increases in hippocampal brain-derived neurotrophic factor (R = 0.46, p<0.01), suggesting a common mechanism for the enhancement of memory. However, unlike protein levels of BDNF, the regional enhancement of BDNF mRNA expression in the hippocampus appeared to be predominantly enhanced by anthocyanins. Our data support the claim that flavonoids are likely causal agents in mediating the cognitive effects of flavonoid-rich foods.

Figure 1. Effect of 6 weeks blueberry (BB), Anthocyanins Extract (A) and Flavanols (F) on spatial working memory in aged rats (18 months old).

A) Effect of flavonoid-rich diets (BB, A or F) on correct choices (± standard error of the mean) in a X-maze Alternation Task: a significant increase in choice accuracy was observed between the control group and the blueberry group (P<0.005); the control and the anthocyanins groups (P<0.01) and the control and the flavanols groups (P<0.005). Maximum score is 8 correct choices (Control group: Triangle ‘down’; Blueberry group: Square; Anthocyanins group: Circle; Flavanols group: Triangle ‘up’). B) Comparison between animals performance at baseline and following 6 week supplementation with either a Control, Blueberry, Anthocyanins or Flavanol diet. * Indicates a significant increase in number of correct choices in comparison to baseline performance, P<0.05.

Figure 2. Levels of brain-derived neurotrophic factor (BDNF) in the hippocampus.

A) Dissected hippocampal tissue lysates were probed for levels of pro-BDNF and BDNF using antibodies that detect the pro-domain of the BDNF protein and the mature protein. Representative immunoblots showing protein levels in 2 animals on the control diet (C), 2 animals supplemented with 2% BB diet (BB), 2 animals supplemented with Anthocyanins Extract (A) and 2 animals supplemented with Flavanols ((−) Epicatechin and (+) Catechin) (F) are presented. Pro-BDNF (grey bars) and mature BDNF (white bars). * Indicates a significant increase in BDNF levels in Blueberry and Anthocyanins groups relative to the control group, P<0.05; n = 6. ** Indicates a significant increase in BDNF levels in Flavanols group relative to control, P<0.01, n = 6. Pro-BDNF and mature BDNF were normalized against GAPDH. B) Correlation between choice accuracy (number of correct choices) in spatial memory task after 6 weeks of dietary interventions (C, BB, A and F) and levels of hippocampal BDNF protein levels, n = 24.

Figure 3. Effects of blueberry supplementation in BDNF mRNA levels in the hippocampus and cortex.

A) Dentate Gyrus (DG) (white bars) and Polymorphic Cell Layer (PCL) (grey bars) of the hippocampus, B) Cortex, C) CA1 D) CA3. Representative pictures of hippocampal and cortical sections showing BDNF mRNA expression from 1 animal from the control (C) group, one from the BB group (BB), one from the Anthocyanins group (A) and one from the Flavanols group (F) are presented. * Indicates a significant increase in BDNF mRNA levels in the anthocyanins group in comparison to control in DG, PCL, CA1 and CA3, P<0.05. No significant differences between the four diet groups were observed in the cerebral cortex. Optical density levels are shown as mean ± SEM derived from at least 6 animals per group. Representative Rnase treated sections are presented for each hippocampal region. The scale presented represents 100 µm.