Electroacupuncture promotes the survival and synaptic plasticity of hippocampal neurons and improvement of sleep deprivation-induced spatial memory impairment

Abstract

Aims: This study aimed to investigate whether electroacupuncture (EA) promotes the survival and synaptic plasticity of hippocampal neurons by activating brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase (TrkB)/extracellular signal-regulated kinase (Erk) signaling, thereby improving spatial memory deficits in rats under SD.

Methods: In vivo, Morris water maze (MWM) was used to detect the effect of EA on learning and memory, at the same time Western blotting (WB), immunofluorescence (IF), and transmission electron microscopy (TEM) were used to explore the plasticity of hippocampal neurons and synapses, and the expression of BDNF/TrkB/Erk signaling. In vitro, cultured hippocampal neurons were treated with exogenous BDNF and the TrkB inhibitor K252a to confirm the relationship between BDNF/TrkB/Erk signaling and synaptic plasticity.

Results: Our results showed that EA mitigated the loss of hippocampal neurons and synapses, stimulated hippocampal neurogenesis, and improved learning and memory of rats under SD accompanied by upregulation of BDNF and increased phosphorylation of TrkB and Erk. In cultured hippocampal neurons, exogenous BDNF enhanced the expression of synaptic proteins, the frequency of the postsynaptic currents, and the phosphorylation of TrkB and Erk; these effects were reversed by treatment with K252a.

Conclusions: Electroacupuncture alleviates SD-induced spatial memory impairment by promoting hippocampal neurogenesis and synaptic plasticity via activation of BDNF/TrkB/Erk signaling, which provided evidence for EA as a therapeutic strategy for countering the adverse effects of SD on cognition.

Keywords: brain-derived neurotrophic factor; electroacupuncture; hippocampal neuron; memory impairment; sleep deprivation; synaptic plasticity.

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

© 2021 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.

Figures

FIGURE 1

EA alleviates spatial memory impairment induced by SD. (A) The location of acupoints (EX‐HN1) in EA treatment. (B, C) Latency to find the platform (B) and swimming speed (C) in the MWM test. (n = 6/group, data were presented as the mean ± SEM and analyzed by nonparametric test (Kruskal‐Wallis test), *p < 0.05 indicates significant difference from the SD group. #p < 0.05 indicates significant difference from the EA + SD group) (D–F) Number of platform crossings (D), swimming speed (E), and time in the target quadrant (F) in the probe test. (n = 6/group, data were presented as the mean ± SEM and analyzed by least significant difference test (LSD),* p < 0.05, ** p < 0.01)(G) Trace plot from the probe test without a hidden platform

FIGURE 2

EA enhances neuron survival in the hippocampus and preserves neurogenesis under SD. (A) Representative images of Nissl staining in different regions of the hippocampus. Scale bar, 100 µm. (B) Schematic illustration of different regions of the hippocampus. Scale bar, 50 µm. (C) Quantification of neurons in the hippocampus. (n = 5/group, data were presented as the mean ± SEM and analyzed by least significant difference test (LSD), *p < 0.05, **p < 0.01). (D) Colocalization of DCX (red) and BrdU (green) in the hippocampus. Scale bar, 50 µm. Boxed areas show higher magnification views. (E) Quantification of DCX+ cells in the DG region of the hippocampus. (n = 5/group, data were presented as the mean ± SEM and analyzed by least significant difference test (LSD), *p < 0.05, **p < 0.01). (F) Percentage of BrdU + DCX + /DCX + cells. (n = 5/group, data were presented as the mean ± SEM and analyzed by least significant difference test (LSD), *p < 0.05, **p < 0.01)

FIGURE 3

EA increases the expression of synaptic proteins in the hippocampus. (A) Western blot analysis of SYP and PSD95 levels in the hippocampus, with β‐actin as the loading control. (B) Quantification of SYP and PSD95 levels from the immunoblot experiment in panel A.( n = 5/group, data were presented as the mean ± SEM and analyzed by least significant difference test (LSD), *p < 0.05, **p < 0.01). (C) Colocalization of SYP (green) and the neuron marker NeuN (red) in the hilus of the hippocampus; nuclei were labeled with Hoechst (blue). Scale bar, 50 µm. (D) Relative density of SYP in the hilus of the hippocampus. (n = 5/group, data were presented as the mean ± SEM and analyzed by least significant difference test (LSD), *p < 0.05, **p < 0.01). (E) Colocalization of PSD95 (green) and NeuN (red) in the hilus; nuclei were labeled with Hoechst (blue). Scale bar, 50 µm. (F) Relative expression of PSD95 in the hilus of the hippocampus. (n = 5/group, data were presented as the mean ± SEM and analyzed by least significant difference test (LSD), *p < 0.05, **p < 0.01)

FIGURE 4

EA prevents and alleviates damage to synapse structure under SD. (A) TEM images of synapses in the hippocampus. Scale bar, 10 µm in panels A–C; 200 nm in panels A1–C1. Boxed areas show higher magnification views of presynaptic (yellow) and postsynaptic (red) terminals. (D–E) Quantitative analysis of synapse number (D), length of active zone (E), and PSD thickness (F) (n = 5/group, data were presented as the mean ± SEM and analyzed by least significant difference test (LSD) or nonparametric test (Kruskal‐Wallis test) (D: LSD test, E: Kruskal‐Wallis test, F: LSD test),*p < 0.05, **p < 0.01)

FIGURE 5

EA enhances BDNF/TrkB/Erk signaling in the hippocampus under SD. (A) Western blot analysis of BDNF, TrkB, p‐TrkB, ERK, and p‐ERK levels in the hippocampus, with β‐actin as the loading control. (B) Quantification of BDNF/β‐actin, p‐TrkB/TrkB, and p‐ERK/ERK levels from the immunoblot experiment in panel A. (n = 5/group, data were presented as the mean ± SEM and analyzed by one‐way ANOVA, *p < 0.05, **p < 0.01). (C–D) Colocalization of BDNF (red) and NeuN (green), p‐TrkB (red) and NeuN (green), and p‐ERK (red) and NeuN (green) in the DG of the hippocampus; nuclei were labeled with Hoechst (blue). Scale bar, 50 µm. (F–H) Percentages of BDNF + NeuN + /NeuN+,p‐TrkB + NeuN + /NeuN, and p‐ERK + NeuN + /NeuN + neurons. (n = 5/group, data were presented as the mean ± SEM and analyzed by nonparametric test (Kruskal‐Wallis test), *p < 0.05, **p < 0.01)

FIGURE 6

Exogenous BDNF enhances the expression of synaptic proteins and TrkB and ERK phosphorylation in primary hippocampal neurons. (A–C) Western blot analysis of SYP, PSD95, TrkB, p‐TrkB, Erk, and p‐Erk levels, with β‐actin as the loading control. (D) Quantification of SYP and PSD95 levels from the immunoblot experiment in panels A–C. (E) Western blot analysis of p‐TrkB/TrkB and p‐Erk/Erk levels. (n = 5/group, data were presented as the mean ± SEM and analyzed by least significant difference test (LSD), *p < 0.05). (F, H) Colocalization of SYP and PSD95 (red) with Tuj‐1 (green) (F) and of p‐TrkB and p‐Erk (red) with Tuj‐1 (green) (H); nuclei were labeled with Hoechst (blue). Scale bar 50 µm. (G, I) Relative densities of SYP/Tuj‐1 and PSD95/Tuj‐1 (G) and relative densities of p‐TrkB/Tuj‐1 and p‐Erk/Tuj‐1(I) (n = 5/group, data were presented as the mean ± SEM and analyzed by least significant difference test (LSD) or nonparametric test (Kruskal‐Wallis test) (SYP/Tuj‐1:LSD test, PSD95/Tuj‐1: Kruskal‐Wallis test, p‐TrkB/Tuj‐1: LSD test, p‐Erk/Tuj‐1: Kruskal‐Wallis test), *p < 0.05, **p < 0.01)