Plant-derived flavanol (-)epicatechin mitigates anxiety in association with elevated hippocampal monoamine and BDNF levels, but does not influence pattern separation in mice

Abstract

Flavanols found in natural products such as cocoa and green tea elicit structural and biochemical changes in the hippocampus, a brain area important for mood and cognition. Here, we evaluated the outcome of daily consumption of the flavanol (-)epicatechin (4 mg per day in water) by adult male C57BL/6 mice on measures of anxiety in the elevated plus maze (EPM) and open field (OF). Furthermore, pattern separation, the ability to distinguish between closely spaced identical stimuli, considered to be mediated by the hippocampal dentate gyrus (DG), was tested using the touchscreen. To investigate mechanisms through which (-)epicatechin may exert its effects, mice were injected with bromodeoxyuridine (50 mg kg(-1)) to evaluate adult hippocampal neurogenesis. In addition, monoaminergic and neurotrophin signaling pathway proteins were measured in tissue derived from subject cortices and hippocampi. Flavanol consumption reduced anxiety in the OF and EPM. Elevated hippocampal and cortical tyrosine hydroxylase, downregulated cortical monoamine oxidase-A levels, as well as increased hippocampal brain-derived neurotrophic factor (BDNF) and pro-BDNF support the flavanol's anxiolytic effects. In addition, elevated pAkt in hippocampus and cortex was observed. (-)Epicatechin ingestion did not facilitate touchscreen performance or DG neurogenesis, suggesting a non-neurogenic mechanism. The concurrent modulation of complementary neurotrophic and monoaminergic signaling pathways may contribute to beneficial mood-modulating effects of this flavanol.

Figure 1

Pattern separation and adult hippocampal neurogenesis. (a) Timeline of the experiments (TS, touchscreen; TT, task training; PT, probe trials; B, BrdU injections; OF, open field; EPM, elevated plus maze; w, weeks of age). (b) The touchscreen paradigm acquisition of big and small separation between two identical stimuli was tested in control (CON) and (−)epicatechin (EC)-treated mice, which were trained to reach a criterion where seven of eight trials were performed correctly. Pattern separation did not differ between the CON and EC groups when the separation between the stimuli was either large or small. (c) Bromodeoxyuridine (BrdU)-positive cell number did not differ between the groups. (d) The percentage of new DG neurons showed no differences between CON and EC-treated mice. Photomicrographs of new cells surviving 9 weeks after the last BrdU injection, in tissue derived from (e) CON and (f) EC-treated subjects. Neuronal phenotype was determined by double-labeling for BrdU (green) and NeuN (red) in (g) CON and (h) EC-treated mice. Error bars denote s.e.m.

Figure 2

Behavioral and cellular assays relevant to mood. (a–c) Anxiety was assayed in the open field (OF) and the elevated plus maze (EPM). (a) Animals were placed inside the OF chamber for 30 min following 7 weeks of (−)epicatechin (EC) consumption. The ratio of time spent and of distance (DIST) travelled by EC subjects in the center of the chamber, relative to the periphery, was significantly higher than that of controls (CON). (b) Total distance travelled in the OF did not differ between the groups. (c) In the EPM, subjects consuming EC displayed no preference between the open or closed arms, whereas CON animals spent a significantly greater amount of time in the closed arms than in the open arms. (d and e) Hippocampi and cortices were assayed for tyrosine hydroxylase (TH), monoamine oxidase (MAO)-A and MAO-B. (d) EC-consuming animals evinced significantly greater hippocampal expression of TH. (e) Increased expression of TH and a reduction in MAO-A was observed in the cortices of EC-supplemented animals (*P<0.05). GADPH, glyceraldehyde-3-phosphate dehydrogenase. Error bars denote s.e.m.

Figure 3

Neurotrophin brain-derived neurotrophic factor (BDNF) and related signaling pathway proteins. (a and b) Immunoblotting of hippocampal and cortical tissue derived from the behaviorally tested animals. (a) (−)Epicatechin (EC)-consuming animals evinced significantly greater expression of pro-BDNF and BDNF proteins in the hippocampus. (b) Growth factor levels did not change in the cortex. (c and d) EC increased the expression of pAkt in the hippocampus and cortex with respect to the control group. (e and f) Neither pERK2 nor pCREB levels were modified by the intake of EC in the hippocampus and cortex. (*P<0.05). Error bars denote s.e.m.