Electroacupuncture stimulation of auricular concha region improves loss of control over stress induced depression-like behavior by modulating 5-hydroxytryptamine 1A receptor

Abstract

Objective: To observe whether electroacupuncture stimulation of auricular concha region (EA-ACR) on behavior changes of depression by loss of control over stress model (LOC), and whether its effect is improved by regulating the expression levels of hydroxytryptamine (serotonin, 5-HT) 1A receptor (5-HT_1AR)/ hydroxytryptamine (serotonin, 5-HT) 2A receptor (5-HT_2AR) in hippocampus.

Methods: LOC was prepared using a Skinner box, and EA-ACR to observe behavioral changes, and Western Blot was used to detect the changes of 5-HT_1AR/5-HT_2AR in the hippocampus, and then observe the changes of EA-ACR behavior after microinjection of 5-HT_1AR/5-HT_2AR antagonist into the hippocampus.

Results: EA-ACR improve depressive-like behavior, up-regulated 5-HT_1AR expression and down-regulated 5-HT_2AR expression in hippocampal brain area. EA-ACR did not improve depression-like behavior after hippocampal microinjection of 5-HT_1AR antagonist, while injection of 5-HT_2AR antagonists can improve depression-like behaviors.

Conclusion: EA-ACR can improve depressive-like behaviors. Loss of control over stress leads to up-regulation of 5-HT_1AR and down-regulation of 5-HT_2AR in the hippocampus, while EA-ACR mainly improves depressive behavior by regulating 5-HT_1AR in Hip.

Keywords: auricular concha region; electroacupuncture stimulation; hippocampus; loss of control over stress; receptors, serotonin.

Figure 1. Effects of EA-ACR stimulation on depression-like behavior in LOC mice

A: the accumulative shock time during Days 2-7 from the mice of LOC group; B: total number of nosepoker touches during Days 2-7, there was a significant difference in the number of times the nosepoker was touched between the left and right sides; C: there were no observable differences of total distance in the four groups; D: time in the center of the open field; E: time in the darkroom of the Light-dark transition test; F: immobile time of the forced swimming. Control: days 2-4: Ms (0 mA, ≤ 1 min), Enp-off, 30-60s Ri, 5B × 10 s, 10 min Rb; days 5-7: 5b × 10 min us, Enp-invalid; days 8-12 EA-ACR (0 mA/0 Hz) × 20 min × 5 d.; LOC: days 2-4: Ms (0.15 mA, ≤ 1 min), Enp-off, 30-60 s Ri, 5B × 10 s, 10 min Rb; days 5-7: 5b × 10 min us, Enp-invalid; days 8-12 EA-ACR (0 mA/0 Hz) × 20 min × 5 d; Yoked: days 2-7 received the same foot shock intensity and pattern as the LOC, but without control over the shock; days 8-12 EA-ACR (0 mA/0 Hz) × 20 min × 5 d; LOC + EA-ACR: days 2-4: Ms (0.15 mA, ≤ 1 min), Enp-off, 30-60 s Ri, 5B × 10 s, 10 min Rb. days 5-7: 5b × 10 min us, Enp-invalid. days 8-12 EA-ACR (1 mA/2 Hz) × 20 min × 5 d. EA: electroacupuncture; ACR: auricular concha region; LOC: loss of control over stress model; Ms: (0.15 mA, ≤ 1 min) - mild shock (0.15 ma, up to 1 min); Enp-off: effective nose-poke turns off shock 30-60 s; Ri: rest interval (30-60 s), 5B × 10 s: 5 blocks of 10 shocks; 10 min Rb: 10-minute rest between blocks. Statistical analyses were measured using one-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons. Data are presented as mean ± standard error of mean (n = 11). Compared with control group, ^aP < 0.01; compared with yoked group, ^bP < 0.01; compared with LOC group, ^cP < 0.01.

Figure 2. Effect of EA-ACR on 5-HT_1AR and 5-HT_2AR protein expression in Hip of LOC mice

A1: representative Western blot of 5-HT_1AR protein expression in the hippocampus; A2: Western blot analysis of the EA-ACR effect 5-HT_1AR protein expression with β-actin as loading control among different groups; B1: representative Western blot of 5-HT_2AR protein expression in the hippocampus; B2: western blot analysis of the EA-ACR effect 5-HT_2AR protein expression with β-actin as loading control among different groups. Control: days 2-4: Ms (0 mA, ≤ 1 min), Enp-off, 30-60s Ri, 5B × 10 s, 10 min Rb. days 5-7: 5b × 10 min us; days 8-12 EA-ACR (0 mA/0 Hz) × 20 min × 5 d. Enp-invalid; LOC: days 2-4: Ms (0.15 mA, ≤ 1 min), Enp-off, 30-60 s Ri, 5B × 10 s, 10 min Rb; days 5-7: 5b × 10 min us; days 8-12 EA-ACR (0 mA/0 Hz) × 20 min × 5 d; LOC+ EA-ACR: days 2-4: Ms (0.15 mA, ≤ 1 min), Enp-off, 30-60s Ri, 5B × 10s, 10 min Rb; days 5-7: 5b × 10 min us; days 8-12 EA-ACR (1 mA/2 Hz) × 20 min × 5 d. 5-HT_1AR: hydroxytryptamine 1A receptor; LOC: loss of control over stress model; EA: electroacupuncture; ACR: stimulation of auricular concha region; Ms: (0.15 mA, ≤ 1 min) - mild shock (0.15 ma, up to 1 min); Enp-off: effective nose-poke turns off shock 30-60s; Ri: rest interval (30-60 s), 5B ×10 s: 5 blocks of 10 shocks; 10 min Rb: 10-minute rest between blocks. Statistical analyses were measured using one-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons. Data are presented as mean ± standard error of mean (n = 4). Compared with control group, ^aP < 0.01; compared with LOC group, ^bP < 0.01.

Figure 3. WAY-100635 antagonizes 5-HT1AR and EA-ACR does not improve LOC-induced depression-like behaviors

A: time in the center of the open field; B: time in the darkroom of the Light-dark transition test; C: immobile time of the Forced swimming; D: time in the center of the open field; E: time in the darkroom of the Light-dark transition test; F: immobile time of the forced swimming. WAY-100635: LOC + 0.5 ng/nL 150 ng per side N-[2-[4-(2-methoxyphenyl)piperazin-1-yl] ethyl]-N- pyridin-2-ylcyclo hexane carboxamide (150 ng/side); M100907: LOC + 1 ng/nL 300ng per side (R-(+)-a- (2,3-dimethoxyphenil)-1-[4- fluorophenylethyl)]-4-piperidinemethanol (300 ng/side); WAY-100635 + EA-ACR: LOC + 0.5 ng/nL 150 ng per side WAY-100635 + EA-ACR; M100907+EA-ACR: LOC + 1 ng/nL 300 ng per side M100907 + EA-ACR; Saline: 300 nL 0.9%Nacl; Saline + EA-ACR: 300 nL 0.9%Nacl + EA-ACR. Control: control of non-foot-shock; LOC: loss of control over stress model; EA: electroacupuncture; ACR: stimulation of auricular concha region. Statistical analyses were measured using one-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons; Data are presented as mean ± standard error of mean (n = 8). Compared with WAY-100635 group, ^aP < 0.01; compared with WAY-100635 + EA-ACR group, ^bP < 0.01; compared with saline group, ^cP < 0.01; Compared with M100907 group, ^dP < 0.01; compared with M100907 + EA-ACR group, ^eP < 0.01.

Figure 4. EA-ACR regulates LOC-induced depression-like behavior through 5-HT_1AR

A1: representative Western blot of 5-HT_1AR protein expression in the hippocampus; A2: effect of electrodes on 5-HT_1AR protein expression in Hip of LOC mice after antagonism of 5-HT_2AR; B1: representative Western blot of 5-HT_2AR protein expression in the hippocampus; B2: Effect of electrodes on 5-HT_2AR protein expression in Hip of LOC mice after antagonism of 5-HT_1AR. WAY-100635: LOC + 0.5 ng/nL 150 ng per side N-[2-[4-(2-methoxyphenyl)piperazin-1-yl] ethyl]-N-pyridin-2-ylcyclo hexane carboxamide (150 ng/side); M100907: LOC + 1 ng/nL 300 ng per side (R-(+)-a- (2,3-dimethoxyphenil)-1-[4- fluorophenylethyl)]-4- piperidinemethanol (300 ng/side); WAY-100635 + EA-ACR: LOC + 0.5 ng/nL 150 ng per side WAY-100635 + EA-ACR; M100907+EA-ACR: LOC + 1 ng/nL 300 ng per side M100907 + EA-ACR; Saline: 300 nL 0.9%Nacl; Saline + EA-ACR: 300 nL 0.9%Nacl + EA-ACR. Control: control of non-foot-shock; LOC: loss of control over stress model; EA: electroacupuncture; ACR: stimulation of auricular concha region. Statistical analyses were measured using one-way analysis of variance followed by post hoc Bonferroni correction for multiple comparisons; Data are presented as mean ± standard error of mean (n = 4). Compared with control group, ^aP < 0.01; Compared with LOC group, ^bP < 0.01; compared with saline group, ^cP < 0.01; compared with M100907 group, ^dP < 0.01; compared with WAY-100635 group, ^eP < 0.01; compared with Saline + EA-ACR group, ^fP < 0.01.