Acupuncture Alleviates Anxiety and 22-kHz Ultrasonic Vocalizations in Rats Subjected to Repeated Alcohol Administration by Modulating the Brain-Derived Neurotrophic Factor/Corticotropin-Releasing Hormone Signaling Pathway

Abstract

The Shenmen point (acupuncture point heart 7: HT7), located in the heart meridian, is frequently used to treat mental disorders, including drug addiction, anxiety, and depression. This study aimed to determine how HT7 regulates anxiety and negative emotions caused by repeated alcohol administration, focusing on the amygdala and paraventricular nucleus (PVN). Repeated administration of alcohol (ETOH; 2 g/kg, i.p. injection, 16% v/v) for 14 days increased the corticosterone (CORT) levels, and HT7 stimulation reduced the plasma CORT levels. HT7 stimulation mitigated anxiety-like behaviors and reduced 22-kHz ultrasonic vocalizations in rats receiving repeated ETOH injections. HT7 stimulation increased the amygdala expression of mature brain-derived neurotropic factor (mBDNF) and phosphorylated tropomyosin receptor kinase B (pTrkB) and decreased the PVN corticotropin-releasing hormone (CRH) expression. Amygdala microinjections of the TrkB antagonist ANA-12 (0.1 pmol/1 μL) reversed the increase in PVN CRH levels. The reduced PVN CRH levels were regulated by CRH-expressing neurons in the amygdala, and the increased amygdala CRH levels were affected by the HT7-stimulation induced increases in mBDNF. HT7 stimulation alleviates increased stress hormone levels and mitigates anxiety and negative emotions caused by repeated ETOH administration. These results provide scientific support for the clinical use of acupuncture to treat various alcoholism-induced diseases.

Keywords: BDNF; CRH; USVs; acupuncture; alcohol.

Figure 1

Schematic of the experimental procedure for acute and repeated ETOH administration (a). Weight changes were monitored for 13 days (n = 4–5 rats per group) (b). Plasma ETOH levels following acute and repeated (14 days) ETOH administration (n = 4–6 rats per group) (c). Plasma corticosterone (CORT) levels (n = 6–8 rats per group) (d). The data were analyzed using repeated measures one- and two-way ANOVA and t-tests. * p < 0.05 compared with the acute Sal group and ^# p < 0.05 compared with the repeated Sal group. Values are presented as means ± SEM.

Figure 2

Schematic of the experimental procedure for repeated ETOH administration and representation of the mechanical acupuncture instrument (MAI) treatment (a). Plasma ETOH levels after acupuncture stimulation (n = 4–6 rats per group) (b). Plasma CORT levels (n = 7–9 rats per group) (c). The data were analyzed using repeated measures one- and two-way ANOVA and t-tests. * p < 0.05 compared with the Sal + Veh group and ^# p < 0.05 compared with the ETOH + Veh group. Values are presented as means ± SEM.

Figure 3

Schematic showing the acupuncture stimulation and elevated plus maze (EPM) test schedule (a). Quantification of the number of entries into the closed arms (b), open arms (c), and center (d) of the EPM (n = 10–11 rats per group). Anxiety indexes in the EPM (e). The data were analyzed using repeated measures ANOVA followed by Tukey’s test. * p < 0.05 compared with the Sal group and # p < 0.05 compared with the ETOH group. Values are presented as means ± SEM.

Figure 4

Schematic showing the acupuncture stimulation and schedule for the measurement of ultrasonic vocalizations (USVs) (a). Number of 22-kHz USVs emitted during the treatment sessions for 20 min after MAI stimulation (n = 7–9 rats per group) (b). The data were analyzed using repeated measures ANOVA followed by Tukey’s test. * p < 0.05 compared with the Sal group and # p < 0.05 compared with the ETOH group. Values are presented as means ± SEM.

Figure 5

Schematic showing the acupuncture stimulation and sacrifice schedule (a). Representative micrographs showing the expression of brain-derived neurotrophic factor (BDNF) in the amygdala. The scale bar represents 100 μm. The results are presented as the number of mature BDNF (mBDNF)-immunoreactive cells (n = 5–7 rats per group) (b). The levels of pro-BDNF in the amygdala (n = 46 rats per group) (c). The levels of mature BDNF in the amygdala (n = 4–6 rats per group) (d). The levels of p75NTR in the amygdala (e). The levels of pTrkB in the amygdala (f). The data were analyzed using repeated measures ANOVA followed by Tukey’s test. * p < 0.05 compared with the Sal group and # p < 0.05 compared with the ETOH group. Values are presented as means ± SEM.

Figure 6

Schematic showing the acupuncture stimulation and sacrifice schedule (a). Representative micrographs showing the expression of corticotropin-releasing hormone (CRH) in the paraventricular nucleus (PVN). The scale bar represents 100 μm (b). The results are presented as the number of CRH-immunoreactive cells (n = 5–7 rats per group) (c). CRH levels in the PVN (n = 4–6 rats per group) (d). The data were analyzed using repeated measures ANOVA followed by Tukey’s test. * p < 0.05 compared with the Sal group, # p < 0.05 compared with the ETOH group, and & p < 0.05 compared with the ETOH + HT7 group. Values are presented as means ± SEM.

Figure 7

Schematic showing the microinjection protocol and needle placement in the PVN (left panel). CRH-immunoreactive neurons (magenta) and the infusion site of Alexa Fluor 488-conjugated cholera toxin subunit B (CTB-488, green) in the amygdala are shown (right panel). The scale bar represents 100 μm (a). Schematic showing the schedule of acupuncture stimulation and microinjection into the amygdala (b). CRH levels in the amygdala after pretreatment with the TrkB antagonist (ANA-12, 0.1 pmol/μL) before stimulation of the Shenmen point (acupuncture point heart 7: HT7) (n = 6–8 rats per group) (c). The proposed mechanism underlying the relationship between BDNF and CRH and the effect of acupuncture stimulation (d). The data were analyzed using repeated measures ANOVA followed by Tukey’s test. * p < 0.05 compared with the Sal group, # p < 0.05 compared with the ETOH group, and & p < 0.05 compared with the ETOH + HT7 group. Values are presented as means ± SEM.