A role for hippocampal PSA-NCAM and NMDA-NR2B receptor function in flavonoid-induced spatial memory improvements in young rats

Abstract

The increase in incidence and prevalence of neurodegenerative diseases highlights the need for a more comprehensive understanding of how food components may affect neural systems. In particular, flavonoids have been recognized as promising agents capable of influencing different aspects of synaptic plasticity resulting in improvements in memory and learning in both animals and humans. Our previous studies highlight the efficacy of flavonoids in reversing memory impairments in aged rats, yet little is known about the effects of these compounds in healthy animals, particularly with respect to the molecular mechanisms by which flavonoids might alter the underlying synaptic modifications responsible for behavioral changes. We demonstrate that a 3-week intervention with two dietary doses of flavonoids (Dose I: 8.7 mg/day and Dose II: 17.4 mg/day) facilitates spatial memory acquisition and consolidation (24 recall) (p < 0.05) in young healthy rats. We show for the first time that these behavioral improvements are linked to increased levels in the polysialylated form of the neural adhesion molecule (PSA-NCAM) in the dentate gyrus (DG) of the hippocampus, which is known to be required for the establishment of durable memories. We observed parallel increases in hippocampal NMDA receptors containing the NR2B subunit for both 8.7 mg/day (p < 0.05) and 17.4 mg/day (p < 0.001) doses, suggesting an enhancement of glutamate signaling following flavonoid intervention. This is further strengthened by the simultaneous modulation of hippocampal ERK/CREB/BDNF signaling and the activation of the Akt/mTOR/Arc pathway, which are crucial in inducing changes in the strength of hippocampal synaptic connections that underlie learning. Collectively, the present data supports a new role for PSA-NCAM and NMDA-NR2B receptor on flavonoid-induced improvements in learning and memory, contributing further to the growing body of evidence suggesting beneficial effects of flavonoids in cognition and brain health.

Keywords: BDNF; Flavonoid; Hippocampus; Learning; Memory; NMDR2B; PSA-NCAM.

Fig. 1

Effects of 3 weeks Flavonoid supplementation on spatial memory in young rats. (a) Effect of flavonoid supplementation on acquisition of the Morris Water Maze task was measured as latency to find the platform (sec). There is a significant decrease in escape latency for both 8.7 mg and 17.4 mg of flavonoids in comparison to control for session 2 (**p < 0.01, n = 8), session 3 and 4 (*p < 0.05, n = 8), indicating a faster acquisition of the task by the flavonoid groups. (b) 24 h recall of spatial memory measured as time spent in target quadrant. Only the 17.4 mg flavonoid group showed significantly enhanced recall of the platform location (*p < 0.05, n = 8). The 8.7 mg flavonoid group showed a trend for increase relative to control animals (p = 0.1).

Fig. 2

Levels of PSA-NCAM in the Dentate Gyrus of the Hippocampus. Levels of polysialylated NCAM cell frequency in the dentate gyrus of the hippocampus of adult Wistar Rats after 3 weeks of flavonoid supplementation. Animals treated with 17.4 mg of flavonoids (n = 5) showed a significant increase in the frequency of PSA-NCAM cells compared to animals on the control diet (n = 4) (**p < 0.01). Illustrative qualitative images of PSA immunoreactivity in the dentate granule cell layer/hilar border (GCL) at −5.6 mm with respect to bregma are presented (1 animal from the control group and 1 animal from the 17.4 mg flavonoid group). The green staining indicates the position of the immunostained cells at the infragranular zone and the scale bar represents 50 μm. Cell identification was aided by the use of the nuclear counter-stain propidium iodide (red). Cell counts were standardized to unit area of granule cell layer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Levels of hippocampal NMDA and AMPA receptors. Hippocampal lysates were immunoblotted with antibodies to detect (a) NMDAR2B, NMDAR2A and NMDAR1; (b) AMPA (GluR 1/2/3). * Indicates a significant increase in NMDAR2B of animals supplemented with 8.7 mg of flavonoids relative to animals supplemented with control solution, p < 0.05; n = 6. *** Indicates a significant increase in NMDAR2B of animals supplemented with 17.4 mg of flavonoids relative to control animals, p < 0.001; n = 6. ^# Indicates a trend toward a decrease in levels of NMDAR2A of animals supplemented with 17.4 mg of flavonoids relative to control animals, p = 0.08, n = 6. GAPDH was used as loading control to normalize total levels of NMDAR2B, NMDAR2A, NMDAR1 and GluR1/2/3. Representative blots showing, left to right, protein levels in two control animals, two animals supplemented with 8.7 mg of flavonoids and two animals supplemented with 17.4 mg of flavonoids are presented.

Fig. 4

Levels of hippocampal ERK1/2 and CREB phosphorylation and total levels of hippocampal BDNF, pro-BDNF and TrkB. Hippocampal lysates were immunoblotted with antibodies to detect: (a) ERK1/2 when dually phosphorylated and total ERK. *Indicates a significant increase in phosphorylation levels of pERK1 (44 kb) and pERK2 (42 kb) of animals supplemented with 8.7 mg of flavonoids relative to animals supplemented with the control (p < 0.05, n = 6). (b) phosphorylated CREB (Ser¹³³) and Total CREB. ^# indicates a trend for an increase of 17.4 mg flavonoid dose in relation to the control solution (p < 0.1, n = 6). CREB and ERK phosphorylation were normalized against total levels of CREB and ERK respectively. (c) Total levels of pro-BDNF (grey bars) and mature BDNF (white bars). * Indicates a significant increase in total levels of BDNF of animals supplemented with 17.4 mg of flavonoids in relation to control animals (p < 0.05, n = 6). (d) Total levels of TrkB (90 kb, 140 kb). GAPDH was used as loading control to normalize total levels BDNF, pro-BDNF and TrkB. Representative blots showing, left to right, protein levels in two control animals, two animals supplemented with 8.7 mg of flavonoids and two animals supplemented with 17.4 mg of flavonoids are presented.

Fig. 5

Levels of hippocampal AKT and mTOR phosphorylation and Arc/Arg3.1. Hippocampal lysates were immunoblotted with antibodies to detect: (a) Akt when phosphorylated at Ser⁴⁷³ and total levels of Akt. ** Indicates a significant increase in phosphorylation levels of Akt in 17.4 mg supplemented animals relative to animals supplemented with a control solution (p < 0.01, n = 6). # Indicates a trend toward increase for animals supplemented with 8.7 mg of flavonoids (p ≤ 0.1, n = 6); (b) mTOR when phosphorylated at Ser ²⁴⁴⁸ (grey bars) and at Ser ²⁴⁸¹ (white bars). * Indicates a significant increase in phosphorylation levels of mTOR at Ser²⁴⁴⁸ in 17.4 mg supplemented animals relative to animals supplemented with control solution (p < 0.05, n = 6); Akt and mTOR phosphorylation were normalized against total levels of Akt and mTOR respectively. (c) Total levels of Arc/Arg3.1. *Indicates a significant increase in total levels of Arc/Arg3.1 in 17.4 mg flavonoid supplemented animals relative to animals supplemented with control solution (p < 0.05, n = 6); # Indicates a trend towards an increase for animals supplemented with 8.7 mg of flavonoids (p ≤ 0.1, n = 6). GAPDH was used as loading control to normalize total levels of Arc/Arg3.1. Representative blots showing, left to right, protein levels in two control animals, two animals supplemented with 8.7 mg of flavonoids and two animals supplemented with 17.4 mg of flavonoids are presented.