Dietary Levels of Pure Flavonoids Improve Spatial Memory Performance and Increase Hippocampal Brain- Derived Neurotrophic Factor Catarina Rendeiro 1,2 , David Vauzour 1¤ , Marcus Rattray 3 , Pierre Waffo-Te ´ guo 4 , Jean Michel Me ´ rillon 4 , Laurie T. Butler 2 , Claire M. Williams 2 , Jeremy P. E. Spencer 1 * 1 Molecular Nutrition Group, School of Chemistry, Food and Pharmacy, University of Reading, Reading, United Kingdom, 2 School of Psychology and Clinical Language Sciences, University of Reading, Reading, United Kingdom, 3 Reading School of Pharmacy, University of Reading, Reading, United Kingdom, 4 University de Bordeaux, ISVV, Groupe d’Etude des Substances Ve ´ge ´tales a ` Activite ´ Biologique, Villenave d’Ornon, France 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. Citation: Rendeiro C, Vauzour D, Rattray M, Waffo-Te ´ guo P, Me ´rillon JM, et al. (2013) Dietary Levels of Pure Flavonoids Improve Spatial Memory Performance and Increase Hippocampal Brain-Derived Neurotrophic Factor. PLoS ONE 8(5): e63535. doi:10.1371/journal.pone.0063535 Editor: Jose Vina, University of Valencia, Spain Received November 30, 2012; Accepted April 3, 2013; Published May 28, 2013 Copyright: ß 2013 Rendeiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Biotechnology and Biological Sciences Research Council (grant BB/F008953/1) and the Fundac ¸a ˜ o para a Cie ˆ ncia e a Tecnologia (FCT) (grant SFRH/BD/69711/2010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: j.p.e.spencer@reading.ac.uk ¤ Current address: Department of Nutrition, Norwich Medical School, Faculty of Medicine and Health Sciences, University of East Anglia, Norwich, United Kingdom Introduction Phytochemical–rich foods, particularly those rich in flavonoids, have been shown to be effective in reversing age-related deficits in memory and learning [1–6]. In particular, studies using Camellia sinensis (tea) [7–12], Gingko Biloba [13–15], Theobroma cacao (cocoa) [16–18] and Vaccinium spp (blueberry) [19–23] have demonstrated beneficial effects on memory and learning in both humans and animal models. Whilst these studies clearly demonstrate the efficacy of flavonoid-rich foods in promoting cognitive perfor- mance, they fall short of providing evidence that flavonoids themselves are the causal agents in driving beneficial effects on memory, learning and neuro-cognitive performance. Because each of these foods contains large array of macro- and micro-nutrients and a diverse phytochemical profile (flavonoids, hydroxycinna- mates, phenolic acids), to date it has been difficult to assign specific biological functions to a single flavonoids or even specific flavonoid groups. Several studies have indicated that absorbed flavonoids and their metabolites are able to transverse the blood-brain-barrier [24–26] and may exert neuropharmacological actions at the molecular level, influencing signalling pathways, gene expression and protein function [27–30]. For instance, the beneficial effects of green tea, blueberry and Gingko Biloba on spatial memory have been shown to involve increases in hippocampal brain-derived neurotrophic factor (BDNF) [23,31–33]. The conversion of short- term memory (STM) into long-term memory (LTM) is regulated at the molecular level in neurons [34–36] and involves the synthesis of new proteins that control neuronal morphology and connectivity [37]. A growing body of evidence indicates that BDNF plays a key role in the regulation of both short-term synaptic function and long-term activity-dependent synaptic plasticity during memory formation [38–40]. Furthermore, declines in hippocampal BDNF levels occur during aging [41– 43] and appear to negatively impact on memory performance [44], whilst both exercise and diet have been shown to influence BDNF expression in the hippocampus [45,46]. We have previously shown that blueberry intervention induces both spatial memory improvements and BDNF signalling in young and old animals [23,47]. However, despite evidence for the functional and molecular actions of blueberry and other flavonoid- rich foods, limited data exist with regards to the actions of pure flavonoids on memory [48–51]. For example, Maher et al (2006) [51] reported that administration of pure fisetin improves recognition memory in rodents, although the underpinning mechanisms were investigated ex-vivo in hippocampal slices. In PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e63535