
==== Front
CNS Neurosci Ther
CNS Neurosci Ther
10.1111/(ISSN)1755-5949
CNS
CNS Neuroscience & Therapeutics
1755-5930
1755-5949
John Wiley and Sons Inc. Hoboken

10.1111/cns.70035
CNS70035
CNSNT-2024-131.R2
Original Article
Original Article
Involvement of basolateral amygdala‐rostral anterior cingulate cortex in mechanical allodynia and anxiety‐like behaviors and potential mechanisms of electroacupuncture
Chen et al.
Chen Yuerong https://orcid.org/0000-0002-7233-4910
1
Tong Siyuan 1
Xu Yingling https://orcid.org/0009-0008-8335-4715
1 2 3
Xu Yunyun 1 4
Wu Zonglin 1
Zhu Xixiao 1
Wang Xirui https://orcid.org/0000-0003-4484-5390
1
Li Chaoran 1
Lin Chalian 1
Li Xiaoyu 1
Zhang Chi 1
Wang Yifang 1
Shao Xiaomei 1 13185097375@163.com

Fang Jianqiao 1 fangjianqiao@163.com

Wu Yuanyuan https://orcid.org/0000-0001-8884-0802
1 runnaway@126.com

1 Key Laboratory of Acupuncture and Neurology of Zhejiang Province, Department of Neurobiology and Acupuncture Research The Third Clinical Medical College, Zhejiang Chinese Medical University Hangzhou China
2 NHC and CAMS Key Laboratory of Medical Neurobiology, MOE Frontier Science Center for Brain Research and Brain‐Machine Integration, School of Brain Science and Brain Medicine Zhejiang University Hangzhou China
3 Liangzhu Laboratory Zhejiang University Medical Center Hangzhou China
4 Tuina Department Hangzhou Red Cross Hospital Hangzhou China
* Correspondence
Yuanyuan Wu, Jianqiao Fang and Xiaomei Shao, Key Laboratory of Acupuncture and Neurology of Zhejiang Province, The Third Clinical Medical College, Zhejiang Chinese Medical University, 548 Binwen Road, Binjiang District, Hangzhou 310053, China.
Email: runnaway@126.com, fangjianqiao@163.com and 13185097375@163.com

15 9 2024
9 2024
30 9 10.1111/cns.v30.9 e7003524 7 2024
23 1 2024
27 8 2024
© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.
https://creativecommons.org/licenses/by/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Abstract

Aims

Chronic pain is highly associated with anxiety. Electroacupuncture (EA) is effective in relieving pain and anxiety. Currently, little is known about the neural mechanisms underlying the comorbidity of chronic pain and anxiety and the EA mechanism. This study investigated a potential neural circuit underlying the comorbid and EA mechanisms.

Methods

Spared nerve injury (SNI) surgery established the chronic neuropathic pain mouse model. The neural circuit was activated or inhibited using the chemogenetic method to explore the relationship between the neural circuit and mechanical allodynia and anxiety‐like behaviors. EA combined with the chemogenetic method was used to explore whether the effects of EA were related to this neural circuit.

Results

EA attenuated mechanical allodynia and anxiety‐like behaviors in SNI mice, which may be associated with the activity of CaMKII neurons in the basolateral amygdala (BLA). Inhibition of BLACaMKII‐rACC induced mechanical allodynia and anxiety‐like behaviors in sham mice. Activation of the BLACaMKII‐rACC alleviated neuropathic pain and anxiety‐like behaviors in SNI mice. The analgesic and anxiolytic effects of 2 Hz EA were antagonized by the inhibition of the BLACaMKII‐rACC.

Conclusion

BLACaMKII‐rACC mediates mechanical allodynia and anxiety‐like behaviors. The analgesic and anxiolytic effects of 2 Hz EA may be associated with the BLACaMKII‐rACC.

Schematic diagram illustrating the potential mechanism by which the BLACaMKII‐rACC neural circuit mediates mechanical allodynia and anxiety‐like behaviors in SNI mice, as well as the analgesic and anxiolytic effects of EA.

anxiety
basolateral amygdala
chronic pain
electroacupuncture
rostral anterior cingulate cortex
Natural Science Foundation of Zhejiang Province 10.13039/501100004731 LQ24H270003 LY19H270007 LY23H270009 National Natural Science Foundation of China 10.13039/501100001809 82074541 College Students’ Innovative Entrepreneurial Training Plan Program202210344020 Zhejiang Chinese Medical University 10.13039/501100004863 2021YKJ08 2021YKJ09 source-schema-version-number2.0
cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:16.09.2024
Chen Y , Tong S , Xu Y , et al. Involvement of basolateral amygdala‐rostral anterior cingulate cortex in mechanical allodynia and anxiety‐like behaviors and potential mechanisms of electroacupuncture. CNS Neurosci Ther. 2024;30 :e70035. doi:10.1111/cns.70035

The first two authors contributed equally to this work.
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pmc1 INTRODUCTION

Chronic pain causes tremendous burdens, affecting over 30% of people around the world. 1 Patients with chronic pain can suffer from mental disorders, including anxiety. 2 , 3 Currently, the use of selective serotonin reuptake inhibitors (SSRIs) and 5‐serotonin noradrenaline reuptake inhibitors (SNRIs) is beneficial in the treatment of chronic neuropathic pain and mental disorders. 4 , 5 Meanwhile, SSRIs and SNRIs often produce adverse effects such as gastrointestinal effects. 6 , 7 Electroacupuncture (EA), as a safe treatment, can alleviate chronic neuropathic pain and anxiety. 8 , 9 However, the mechanism of EA is still not fully elucidated.

The amygdala is essential for regulating pain and anxiety. 10 Injection of glutamate receptor antagonists into basolateral amygdala (BLA) in mice can alleviate neuropathic pain. 11 Injection of the antidepressant paroxetine into the BLA of neuropathic pain mice reduces anxiety‐related behaviors. 12 Our previous research has indicated BLA is linked to anxiety‐like behaviors induced by spared nerve injury (SNI). 13 Therefore, our study focused on the role of BLA regarding chronic neuropathic pain and anxiety‐like behaviors.

Rostral anterior cingulate cortex (rACC) is involved in neuropathic pain‐related negative emotion. 14 , 15 Besides, glutamatergic (Glu) receptors on the rACC are engaged in the emotional response to pain. 16 Our study showed there are CaMKII neuronal projections from BLA to rACC (BLACaMKII‐rACC). 17 However, it is currently unclear whether BLACaMKII‐rACC is involved in chronic neuropathic pain and anxiety‐like behaviors.

In our previous research, EA mediated BLA to relieve mechanical allodynia and anxiety‐like behaviors. 13 EA regulated rACC to suppress negative emotions. 18 However, it remains unclear whether EA alleviates chronic neuropathic pain and anxiety‐like behaviors via the BLACaMKII‐rACC.

Therefore, this study aims to investigate whether the BLACaMKII‐rACC neural circuit mediates chronic neuropathic pain and anxiety‐like behaviors and whether the analgesic and anxiolytic effects of EA are related to the BLACaMKII‐rACC neural circuit. Chronic neuropathic pain mice prepared by SNI surgery. Activity of CaMKII neurons in BLA detected by immunofluorescence technique. The anatomical connection between BLA and rACC was validated via viral tracing techniques. The activation or inhibition of the BLACaMKII‐rACC neural circuit was manipulated by chemogenetic method. Mechanical allodynia was assessed via paw withdrawal thresholds (PWTs). Both elevated plus maze test (EPMT) and open field test (OFT) were used to observe anxiety‐like behaviors.

2 MATERIALS AND METHODS

2.1 Animals

All experiments mice were adult male C57BL/6J mice (22–25 g, 8–10 w). The mice were provided and raised by the Experimental Animal Center of Zhejiang Chinese Medical University. Ventilation and air filtration units were available for the study. The mice were raised under a 12‐h light/dark cycle (light cycle 8:00 a.m.–8:00 p.m.) at room temperature of 23–25°C and humidity of 40%–60%. Each cage housed 4 male mice with corn cob bedding at the bottom. The mice were fed with standard pellet chow and water ad libitum. The experimental operations complied with the experimental ethics requirements of the Experimental Animal Management and Ethics Committee of Zhejiang Chinese Medical University (IACUC‐20210118‐07) and were conducted following the relevant provisions of the Regulations on the Administration of Laboratory Animals of the People's Republic of China and the spirit of humanitarianism.

2.2 von Frey filament test

The mice were placed on wire mesh and covered with translucent plexiglass cover. They were allowed to acclimate to the environment for 1 h. After the mice were quiet, the mice were stimulated with von Frey filaments in the plantar surface of the left hind paw until the von Frey filaments bent into an S‐shape and held for 6–8 s. A positive reaction was counted when mice retracted, licked, or flinched their claws quickly. Stimulation intervals for von Frey filaments should be at least 1 min each time. The force of the von Frey filament with three positive reactions out of five tests was recorded as PWTs. 19 PWTs were measured at baseline, day 7, and day 14 after modeling. 20

2.3 Chronic neuropathic pain mouse model

SNI surgery was used to establish a chronic neuropathic pain model. 21 Mice were anesthetized with 0.3% pentobarbital sodium (60 mg/kg, ip). The hair of the left hind limb was removed. The skin was disinfected with iodophor and 75% ethanol. An incision was made on the skin above the midpoint between the tibial head and the greater trochanter of the femur. Then, the muscles were bluntly dissected to expose the sciatic nerve. The peroneal nerve and common peroneal nerve were tightly ligated with non‐absorbent 6–0 sutures and the nerves were transected. A section of 2–3 mm was removed distal to the ligature, leaving the tibial nerve intact. The incision was closed in layers and disinfected with iodophor. The sham group underwent the same surgery without the ligation or severance of the nerve.

2.4 Viral injection

Mice were anesthetized with 0.3% sodium pentobarbital (60 mg/kg, ip). The mice were fixed on a stereotaxic frame (RWD, 68025, Shenzhen, China). A glass microelectrode for injection was connected to an infusion pump (WPI, UMC4, Sarasota, FL, United States). A volume of 160 nL of virus was administered into the BLA at a rate of 100 nL/min (BLA: anterior–posterior: −1.36 mm; mediolateral: ±3.20 mm; dorsoventral: −4.25 mm). A volume of 90 nL of virus was injected into the rACC at a rate of 60 nL/min (rACC: anterior–posterior: +1.35 mm; mediolateral: ±0.25 mm; dorsoventral: −0.85 mm). A dental drill (WPI, OmniDrill35, Sarasota, FL, United States) was used to create the cranial openings. At the end of the infusion, the glass microelectrode was left at the injection site for 10 min to prevent the virus from overflowing. The BLA coordinates and rACC coordinates were determined based on Paxinos and Franklin's The Mouse Brain in Stereotaxic Coordinates (Fourth version).

2.5 Tracer virus injection strategy

Mice were injected with anterograde tracer virus (AAV2/9‐CaMKIIα‐EGFP, 5.34 × 1012 vg/mL, BrainVTA, China, PT‐0290) in the right BLA. The virus (AAV2/R‐CaMKIIα‐EGFP, 5.24 × 1012 vg/mL, BrainVTA, PT‐0290) was injected into the right rACC for retrograde monosynaptic tracking. After 2 weeks, the tracer virus successfully transfected neurons in BLA and rACC. Then, the mice were anesthetized with 0.3% sodium pentobarbital (60 mg/kg, ip) and perfused sequentially with saline (20 mL) and 4% paraformaldehyde (20 mL) via the cardiac. Finally, 20 μm brain slices were prepared for recording green fluorescent protein signals.

2.6 Chemogenetic method

The BLACaMKII‐rACC neural circuit was manipulated via the chemogenetic method to determine whether the neural circuit modulated mechanical allodynia and anxiety‐like behaviors. The mice were injected rAAV2/9‐CaMKIIα‐DIO‐hM4Di‐mCherry‐WPRE‐pA (3.38 × 1012 vg/mL, BrainVTA, China, PT‐1143), rAAV2/9‐CaMKIIα‐DIO‐hM3Dq‐mCherry‐WPRE‐pA (3.04 × 1012 vg/mL, BrainVTA, China, PT‐1144) or rAAV2/9‐CaMKIIα‐DIO‐mCherry‐WPRE‐pA (5.35 × 1012 vg/mL, BrainVTA, China, PT‐1167) into the BLA. rAAV2/R‐CaMKIIα‐Cre (6.65 × 1012 vg/mL, BrainVTA, China, PT‐0220) was injected into the rACC. Clozapine‐N‐oxide (CNO) (0.2 mg/mL, dissolved in dimethyl sulfoxide and diluted with 0.9% saline, 2 mg/kg, ip) was intraperitoneally injected on days 8, 10, 12, 14, and 16 after SNI/sham surgery. Among them, rAAV2/9‐CaMKIIα‐DIO‐hM4Di‐mCherry‐WPRE‐pA and rAAV2/R‐CaMKIIα‐Cre were injected bilaterally to inhibit the BLACaMKII‐rACC neural circuit. rAAV2/9‐CaMKIIα‐DIO‐hM3Dq‐mCherry‐WPRE‐pA and rAAV2/R‐CaMKIIα‐Cre were injected into the right side to activate the BLACaMKII‐rACC neural circuit. The rAAV2/9‐CaMKIIα‐DIO‐mCherry‐WPRE‐pA is a control virus without any activating or inhibitory effect.

2.7 EA treatment

EA treatment was conducted on the 8th, 10th, 12th, 14th, and 16th days after SNI surgery. In addition to the EA group, the sham, SNI, and SNI‐sham EA groups were all fixed loosely. Bilateral Zusanli (ST36) and Sanyinjiao (SP6) acupoints were selected. 0.16 × 7 mm acupuncture needles were directly inserted into the acupoints for 5 mm in the EA group. The two needles on the same side were connected to the Hans acupoint nerve stimulator (HANS‐200A, Beijing Hua Wei Industrial Development Co). The intensity of EA was 0.3 mA, the frequency was 2 Hz and the stimulation time was 30 min. In the SNI‐sham EA group, electrodes, but no current, were attached to the acupuncture needles.

2.8 Elevated plus maze test (EPMT)

The EPMT is the most commonly used test for studying general anxiety‐like behavior. 22 Mice were subjected to EPMT on day 14 after sham/SNI surgery. The day before EPMT, the mice were transferred to the behavioral room to acclimatize. The room temperature was 23–25°C and the humidity was 40%–60%. The elevated plus maze (EPM) was composed of two open arms (30 × 6 cm), two closed arms (30 × 6 × 15 cm), and a center area (6 × 6 cm) with a height of 35 cm. The open arms and closed arms are crossed vertically. In the beginning, each mouse was placed in the center area with its head facing the open arm. After the experiment, the urine and feces of the mice were cleaned up. To remove the odor left by the last animal, the EPM will be cleaned with 75% ethanol and double‐distilled water. The mice's behaviors during the test were recorded on a video tracking system (ANY‐maze V6.14, Stoelting, USA). The video was recorded for 5 min and 30 s, with the first 30 s being the acclimatization phase.

2.9 Open field test (OFT)

The OFT was conducted on day 16 after surgery to determine whether mice displayed anxiety‐like behaviors. 23 The day before OFT, the mice were relocated to the behavioral room. The open field (OF) was a 40 × 40 × 40 cm3 uncovered cube. The bottom of the cube was divided equally into 16 squares of identical area. The outer 12 square areas were defined as the peripheral area. The 4 square areas in the middle are defined as the central zone (with a total area of 20 × 20 cm2). The mice were placed in the center zone at the beginning. Finally, the mice's urine and feces were cleaned up. The interior of the OF was cleaned with 75% ethanol and double‐distilled water in turn. The mice's behaviors during testing were recorded on a video tracking system (ANY‐maze V6.14, Stoelting, USA). The duration of the video is 5 min and 30 s.

2.10 Immunofluorescence staining

Approximately 90 min after OFT, mice were deeply anesthetized with sodium pentobarbital (60 mg/kg, ip). The mice were perfused transcardially with 0.9% saline and 4% (w/v) paraformaldehyde sequentially. After perfusion, the mice brains were removed and placed in 4% (w/v) paraformaldehyde for 24 h and then subjected to gradient dehydration with 15% and 30% (w/v) sucrose. The brains were sliced into frozen coronal sections of 20 μm thickness using the freezing microtome (CryoStar NX50 HOP, Thermo Fisher Scientific).

The slices were mounted on gelatin‐coated glass. The slices were rewarmed at 37°C for 1 h, and they were washed with Tris Buffered Saline with Tween‐20 (TBST) on a shaker 6 times for 10 min each. The slices were blocked with 10% donkey serum (with 0.3% Triton X‐100) for 1 h at 37°C and incubated with the primary antibodies for 20 h at 4°C. The primary antibodies included anti‐CaMKII (mouse, 1:200, Abcam, ab22609) and anti‐cFos (rabbit, 1:500, Abcam, ab190289). The slices were then rewarmed again at 37°C for 1 h, washed 6 times, and incubated with Alexa Fluor 647‐conjugated secondary antibody (1:1000, Abcam, ab150111) and Alexa Fluor 488‐conjugated secondary antibody (1:800, Jackson ImmunoResearch Labs, 711‐545‐152) for 1 h at 37°C. The slices were washed again with TBST, followed by incubation with 4′,6‐diamidino‐2‐phenylindole (DAPI, Abcam, USA). The fluorescence signal was then observed using a ZEISS digital scanner (ApoTome.2, ZEISS, Germany).

2.11 Statistical analysis

All analyses and graphs were calculated and plotted using GraphPad Prism 9. All results are expressed as mean ± standard errors of the mean (SEM). The normality of the distribution of continuous variables was assessed using the Shapiro–Wilk normality test. The PWTs between different groups were tested with two‐way repeated‐measures Analysis of Variance (ANOVA) with Tukey's post‐hoc test. For normally distributed data, Student's t‐test (two‐tailed) and one‐way ANOVA followed by Tukey's post‐hoc test were used to compare means of two and multiple groups, respectively. Additionally, the Mann–Whitney U‐test and Kruskal–Wallis were employed to compare two or more groups of non‐normally distributed data, respectively. A significance level of p < 0.05 was considered statistically significant.

3 RESULTS

3.1 2 Hz EA reduced mechanical allodynia and anxiety‐like behaviors in SNI mice

Research findings indicate that EA effectively alleviates mechanical allodynia and anxiety‐like behaviors. 24 , 25 , 26 Therefore, we aim to further investigate the effects of 2 Hz EA on mechanical allodynia and anxiety‐like behaviors in neuropathic pain mice (Figure 1A). We established a chronic neuropathic pain mouse model by employing SNI injury on C57BL/6J mice (Figure 1B), following previously reported protocols. 27 Similar to previous studies, we chose bilateral Zusanli (ST36) and Sanyinjiao (SP6) as the sites for EA stimulation (Figure 1C). 25 , 26

FIGURE 1 2 Hz EA reduced mechanical allodynia and anxiety‐like behaviors in SNI mice. (A) Experimental flow chart. (B) SNI surgery schematic diagram. (C) Schematic diagram of EA intervention. The acupoints for EA intervention were bilateral Zusanli (ST36) and Sanyinjiao (SP6), with a stimulation frequency of 2 Hz, intensity of 0.3 mA, and 30 min. (D) A comparison of PWTs among 4 groups. (E) Time spent in the open arms among 4 groups. (F) Time spent in the center zone among 4 groups. (G) The total distance traveled by the 4 groups. (H) Representative motion trajectories and activity heat maps in EPMT (I) Representative motion trajectories and activity heat maps in OFT. *p < 0.05, ### p < 0.001 compared with SNI group; # p < 0.05, && p < 0.01 compared with SNI‐EA group; & p < 0.05 compared with SNI‐sham EA group; ns, not significant. n = 8–12 (D–G). (D) two‐way ANOVA and Tukey's test; (E–G) One‐way ANOVA and Tukey's test.

Compared to the sham mice, the PWTs in the SNI mice decreased (Figure 1D). The SNI mice spent less time in the open arm and central zone (Figure 1E,F). The result suggests that SNI surgery can induce neuropathic pain and anxiety‐like behaviors in mice.

After EA intervention, the PWTs of the SNI‐EA mice increased compared to SNI mice and SNI‐sham EA mice (Figure 1D). SNI‐EA mice exhibited an increase in open‐arm and central zone time (Figure 1E,F). The results suggest that 2 Hz EA is effective at reducing mechanical allodynia and anxiety‐like behaviors in SNI mice. At the same time, there were no statistically significant differences in the total distance traveled among the 4 groups (Figure 1G), which suggests the locomotor activity of the 4 groups was not affected. Figure 1H,I show the trajectory maps and heat maps of the 4 groups. These results underscore the efficacy of the SNI mice in inducing mechanical allodynia and anxiety‐like behaviors. Moreover, 2 Hz EA significantly mitigated chronic neuropathic pain and anxiety‐like behaviors in SNI mice.

3.2 The effects of EA on mechanical allodynia and anxiety in SNI mice may be linked to BLA CaMKII neurons activity

Research suggests BLA mediates neuropathic pain. 11 , 28 , 29 Our preliminary research indicates BLA is also associated with anxiety‐like behavior induced by SNI. 13 Modulating BLA influences anxiety‐like behaviors in neuropathic pain mice. 10 Therefore, we aim to further explore whether the analgesic and anti‐anxiety effects of EA on SNI mice are associated with the BLA.

cFos is a marker for neuronal activity. When neurons are activated, the expression of cFos is upregulated. 30 As shown in Figure 2A,B, compared to the sham group, the co‐expression rate of cFos in BLA CaMKII neurons is reduced in SNI mice, suggesting that the mechanical allodynia and anxiety‐like behavior in SNI mice may be associated with decreased activity of BLA CaMKII neurons. Meanwhile, compared to the SNI group, the co‐expression rate of cFos in BLA CaMKII neurons is higher in the SNI‐EA group, indicating that the analgesic and anti‐anxiety effects of EA may be linked to the activity of BLA CaMKII neurons.

FIGURE 2 The colocalization of CaMKII and cFos in the BLA. (A) The representative images of the colocalization of CaMKII and cFos in the BLA among 4 groups. The red fluorescence represents CaMKII, the green fluorescence indicates the neuronal activation marker protein cFos and the blue fluorescence represents the nuclear counterstain DAPI. Scale bar: 20 μm. (B) The statistical results of the colocalization counts of CaMKII and cFos in the BLA. **p < 0.01, compared with SNI group; ns, not significant. n = 3 (with 5 brain slices per mouse).

3.3 CaMKII neurons project from BLA to rACC

To investigate whether BLA is structurally related to rACC, an anterograde tracing virus (AAV2/9‐CaMKIIα‐EGFP) was injected into the right BLA (Figure 3A). After 2 weeks, CaMKII neurons that were transfected and labeled by the virus were observed in the right BLA (Figure 3B). In addition, nerve fibers transfected and labeled by the virus could be seen in the right rACC (Figure 3C). Furthermore, we injected retrograde tracer viruses (AAV2/R‐CaMKIIα‐EGFP) into the right rACC (Figure 3D). Two weeks after injection, transfected CaMKII neurons could be visualized in the right rACC (Figure 3E). The CaMKII neurons transfected by the virus could be observed in the right BLA (Figure 3F). Therefore, CaMKII neurons in the BLA project to the rACC.

FIGURE 3 Circuit diagram for the BLACaMKII‐rACC. (A) Schematic diagram of AAV2/9‐CaMKIIα‐EGFP injection in the right BLA. (B) Representative images of right BLA CaMKII neurons projecting to the right rACC in the right BLA and local magnification of the injection site (top right). Scale bar = 200 μm, scale bar =10 μm (top right). (C) Representative images of right rACC fibers projecting from the right BLA. Scale bar = 100 μm (left), scale bar = 10 μm (top right). (D) Schematic diagram of AAV2/R‐CaMKIIα‐EGFP injection in the right rACC. (E) Representative images of the retrograde tracer virus injection site in the right rACC. Scale bar = 100 μm, scale bar = 10 μm (top right). (F) Representative images of the CaMKII neurons in the right BLA retrograding from the right rACC. Scale bar = 200 μm, scale bar = 10 μm (top right).

3.4 Inhibition of BLACaMKII ‐rACC induced mechanical allodynia and anxiety‐like behaviors in sham mice

Our previous research demonstrated the correlation between the activity of BLA CaMKII neurons and chronic neuropathic pain and anxiety‐like behaviors and the existence of the BLACaMKII‐rACC. In addition, rACC is also implicated in chronic neuropathic pain and pain‐related negative emotions. 31 , 32 However, the role of the BLACaMKII‐rACC in chronic neuropathic pain and anxiety‐like behaviors remains unclear. Therefore, we used chemogenetic methods in sham mice to manipulate the BLACaMKII‐rACC neural circuit. We observed alterations in PWTs, open arm time, and central time to investigate if BLACaMKII‐rACC plays a role in mechanical allodynia and anxiety‐like behaviors.

In sham‐hM4D‐CNO group, bilateral injections of rAAV2/9‐CaMKIIα‐DIO‐hM4Di‐mCherry‐WPRE‐pA were administered into the BLA, while bilateral injections of rAAV2/R‐CaMKIIα‐Cre were delivered into the rACC to inhibit bilateral BLACaMKII‐rACC. In sham‐mcherry‐CNO group, the BLA was injected bilaterally with AAV2/9‐CaMKIIα‐DIO‐mCherry‐WPRE‐pA, the rACC was injected bilaterally with rAAV2/R‐CaMKIIα‐Cre to be used as a control group (Figure 4A,B). After 2 weeks, viral expression was observed in BLA (Figure 4C). Approximately 56.0% of the BLA‐projecting neurons labeled with mCherry were immunoreactive for CaMKII (Figure 4D,E). To determine whether the hM4D virus inhibits the activity of BLA CaMKII neurons projecting to rACC, we compared the percentage of colocalization of the virus‐labeled neurons with cFos between the sham‐mCherry‐CNO and sham‐hM4D‐CNO groups (Figure 4F). Results showed that the hM4D virus reduced the activity of BLA CaMKII neurons projecting to rACC from 17.9% to 11.9% (Figure 4G).

FIGURE 4 Inhibition of BLACaMKII‐rACC‐induced mechanical allodynia and anxiety‐like behaviors in sham mice. (A) Experimental flow chart. (B) Chemogenetic virus injection diagram. (C) Image of viral expression on the BLA. Scale bar = 100 μm. (D) Representative images of BLA CaMKII neurons infected by DIO‐mCherry (red) and stained with CaMKII (green). Scale bar = 20 μm. (E) The percentage of colocalization of mCherry and CaMKII (n = 15 slices from 3 mice). (F) Representative images of BLA CaMKII neurons infected by DIO‐mCherry (red) and stained with cFos (green). Scale bar = 20 μm. (G) The percentage of colocalization of mCherry and cFos (n = 15 slices from 3 mice, *p < 0.05, unpaired sample t‐test). (H) The comparison of PWTs. (I) Time spent in the open arms. (J) Time spent in the center zone. (K) The total distance traveled by the 2 groups in the OFT did not differ statistically. (L) Representative motion trajectories and activity heat maps in EPMT. (M) Representative motion trajectories and activity heat maps in OFT. ****p < 0.0001, **p < 0.01, ***p < 0.001, compared with sham‐mCherry‐CNO group; ns, not significant. n = 10–15 (H–K). (H) two‐way ANOVA and Tukey's test; (I–K) unpaired‐sample t‐test.

In comparison to the sham‐mCherry‐CNO group, the sham‐hM4D‐CNO group exhibited significantly lower PWTs (Figure 4H), suggesting that inhibition of BLACaMKII‐rACC induced mechanical allodynia in sham mice. Compared to the sham‐mCherry‐CNO group, the sham‐hM4D‐CNO group spent less time in the open arm and the center zone (Figure 4I,J). There was no notable variance observed in the total distance traveled (Figure 4K), suggesting similar locomotor abilities between the 2 groups. Figure 4L,M depict the representative motion trajectories and heat maps in EMPT and OFT, respectively. Hence, the results suggest inhibition of BLACaMKII‐rACC‐induced mechanical allodynia and anxiety‐like behaviors in sham mice.

3.5 Activation of BLACaMKII ‐rACC attenuated mechanical allodynia and anxiety‐like behaviors in SNI mice

Since the previous result shows the inhibition of BLACaMKII‐rACC in sham mice can induce mechanical allodynia and anxiety‐like behaviors, we next investigated whether mechanical allodynia and anxiety‐like behaviors in SNI mice could be attenuated by activation of BLACaMKII‐rACC (Figure 5A). First, rAAV2/9‐CaMKIIα‐DIO‐hM3Dq‐mCherry‐WPRE‐pA was injected into the right BLA, and AAV‐CaMKIIα‐Cre was injected into the right rACC to activate BLACaMKII‐rACC (Figure 5B,C). Next, we examined the colocalization of CaMKII neurons with cFos (Figure 5D). It turned out that BLA CaMKII neurons projecting to rACC were significantly activated by the hM3D virus (Figure 5E).

FIGURE 5 Activation of the BLACaMKII‐rACC alleviated mechanical allodynia and anxiety‐like behaviors in SNI mice. (A) Experimental flow chart. (B) Chemogenetic virus injection diagram. (C) Representative image of neurons in the BLA transfected and labeled with mCherry virus. Scale bar = 100 μm. (D) Representative images of BLA CaMKII neurons infected by DIO‐mCherry (red) and stained with cFos (green). Scale bar = 20 μm. (E) The percentage colocalization of mCherry and cFos (n = 15 slices from 3 mice, ****p < 0.0001, unpaired sample t‐test). (F) A comparison of PWTs. (G) Time spent in the open arms. (H) Time spent in the center zone. (I) The total distance traveled by the 2 groups. (J) Representative motion trajectories and activity heat maps in EPMT. (K) Representative motion trajectories and activity heat maps in OFT. *p < 0.05, **p < 0.01, compared with the SNI‐hM3D‐Saline group; ns, not significant. n = 8 (F). n = 12–15 (G–I). (F) two‐way ANOVA and Tukey's test, (G–I) unpaired‐sample t‐test.

Compared to the SNI‐hM3D‐Saline group, the PWTs of the SNI‐hM3D‐CNO group were higher (Figure 5F). The PWTs results suggest activation of BLACaMKII‐rACC relieves mechanical allodynia in SNI mice. In addition, SNI‐3D‐CNO mice spent more time in the open arm and center zone compared with the SNI‐hM3D‐Saline group (Figure 5G,H). The total distance was not statistically different between the 2 groups (Figure 5I). Motion trajectory maps and heat maps of the 2 groups in EPMT and OFT are depicted in Figure 5J,K, respectively. Therefore, it can be concluded that activation of BLACaMKII‐rACC can mitigate mechanical allodynia and anxiety‐like behaviors in SNI mice.

3.6 Inhibition of BLACaMKII ‐rACC antagonized the analgesic and anti‐anxiety effects of EA on SNI mice

The above results indicate that EA can alleviate mechanical allodynia and anxiety‐like behaviors in SNI mice, possibly associated with the activity of BLA CaMKII neurons. Besides, BLACaMKII‐rACC is involved in the mechanical allodynia and anxiety‐like behaviors of both sham and SNI mice. However, it remains unclear if the analgesic and anti‐anxiety effects of EA on SNI mice are linked to BLACaMKII‐rACC.

To investigate whether the effects of EA are mediated through the BLACaMKII‐rACC. In the SNI‐hM4D‐CNO‐EA group, bilateral BLA was injected with rAAV2/9‐CaMKIIα‐DIO‐hM4Di‐mCherry‐WPRE‐pA, bilateral rACC was injected with rAAV2/R‐CaMKIIα‐Cre to inhibit the BLACaMKII‐rACC. After administering CNO injection to SNI mice, EA intervention was performed 30 min later (Figure 6A,B), which allowed us to observe whether the effects of EA were influenced when the BLACaMKII‐rACC was inhibited (Figure 6A,B).

FIGURE 6 Analgesic and anxiolytic effects of EA were associated with BLACaMKII‐rACC neural circuit. (A) Experimental flow chart. (B) Chemogenetic virus injection diagram. (C) After intraperitoneal injection of CNO for 30 min, EA intervention was conducted on SNI mice at the acupoints Zusanli (ST36) and Sanyinjiao (SP6) bilaterally. The EA had a frequency of 2 Hz, an intensity of 0.3 mA, and 30 min. (D) A comparison of PWTs. (E) Time spent in the open arm. (F) Time spent in the center zone. (G) The total distance covered by the 3 groups in the OFT showed no statistically significant difference. (H) Representative motion trajectories and activity heat maps in EPMT. (I) Representative motion trajectories and activity heat maps in OFT. Compared to the SNI‐mCherry‐CNO‐EA group, **p < 0.01, ***p < 0.001; ns, not significant. n = 7–10 (C–F). (C) two‐way ANOVA and Tukey's test. (D–F) one‐way ANOVA and Tukey's test.

Compared to the SNI‐mCherry‐CNO group, mice in the SNI‐mCherry‐CNO‐EA group showed higher PWTs (Figure 6C), confirming the analgesic effect of EA. In comparison to the SNI‐mCherry‐CNO‐EA group, the SNI‐hM4D‐CNO‐EA group exhibited notably lower PWTs (Figure 6C). The PWTs result indicates the analgesic effect of EA is antagonized when the BLACaMKII‐rACC is inhibited.

Compared to the SNI‐mCherry‐CNO group, the SNI‐mCherry‐CNO‐EA group spent more time in the open arm and center zone (Figure 6D,E). The SNI‐hM4D‐CNO‐EA group spent less time in both the open arm and center zone compared to the SNI‐mCherry‐CNO‐EA group (Figure 6D,E). Meanwhile, the total distance traveled by the 3 groups was not statistically different (Figure 6F). Figure 6G,H show the trajectory map and heat map of the 3 groups in the EPMT and OFT, respectively.

Therefore, the above results indicate that when the BLACaMKII‐rACC neural circuit is inhibited, the analgesic and anti‐anxiety effects of EA are antagonized. This suggests that the BLACaMKII‐rACC is involved in the analgesic and anti‐anxiety mechanism of EA.

4 DISCUSSION

Chronic pain is a major cause of disability globally, placing a huge burden on society and families. 33 Patients with chronic pain tend to show higher features of co‐morbidity with psychiatric disorders, including anxiety. 34 Clinical studies have found that antidepressant medications are effective in relieving negative emotions but not pain, often causing side effects. 35 , 36 , 37 , 38 , 39 Therefore, it is crucial to further explore the potential mechanisms of pain‐related negative emotion. Our previous study found that EA had a significant alleviating effect on chronic pain and pain‐related negative emotions. 40 So we would like to further explore the potential mechanisms of EA intervention for pain and pain‐related negative emotions.

Acupuncture is a quite safe treatment. 41 , 42 Our previous study found that 2 Hz EA of bilateral Zusanli (ST36) and Sanyinjiao (SP6) alleviated the chronic neuropathic pain and anxiety‐like behaviors, 20 so we did not further screen the frequency and acupoints for EA in this study. In this study, we validated the analgesic and anxiolytic effects of 2 Hz EA on SNI mice. However, the mechanisms underlying the analgesic and anxiolytic effects of EA are still unclear.

The amygdala plays an important role in pain and anxiety regulation. 43 , 44 , 45 As part of the amygdala, the BLA is involved in both hyperalgesia and anxiety. 46 , 47 , 48 , 49 , 50 In our previous study, it was observed that BLA mediates anxiety‐like behaviors related to pain. 13 Glutamate receptor antagonist was found to exert a powerful analgesic effect in BLA. 11 Our study also found the activity of BLA CaMKII neurons was reduced after SNI surgery. It is suggested that mechanical allodynia and anxiety‐like behaviors in SNI mice may be related to the activity of BLA CaMKII neurons.

Moreover, after EA intervention in SNI mice, mechanical allodynia and anxiety‐like behaviors were alleviated, accompanied by an elevation in the activity of BLA CaMKII neurons. Hence, our study focuses on the correlation between the activity of BLA CaMKII neurons and the manifestation of mechanical allodynia and anxiety‐like behaviors.

rACC is crucial to the regulation of emotions. 51 , 52 Activation of N‐methyl‐D‐aspartate receptors in the rACC is required to acquire pain‐related negative emotions. 31 Activation of N‐methyl‐D‐aspartate receptors on rACC inhibits emotional responses to pain. 16 , 53 Our previous findings suggest that the rACC mediates anxiety‐like behaviors associated with both neuropathic pain and inflammatory pain. 25 , 54 , 55 Accordingly, rACC is closely associated with pain and negative emotions.

Therefore, both the BLA and rACC are highly correlated with pain and negative emotions. Our study found that BLA CaMKII neurons project to the rACC. However, it remains uncertain whether the BLACaMKII‐rACC neural circuit plays a role in mediating mechanical allodynia and anxiety‐like behaviors. We first inhibited BLACaMKII‐rACC in sham mice. The results showed mechanical allodynia and anxiety‐like behaviors were induced in sham mice after the BLACaMKII‐rACC was inhibited. To further substantiate the role of the BLACaMKII‐rACC neural circuit, we activated the neural circuit in SNI mice. The results indicated that when the BLACaMKII‐rACC in SNI mice was activated, both mechanical allodynia and anxiety‐like behaviors in SNI mice were alleviated. At the same time, the activity of the BLA CaMKII neurons that projected to the rACC was elevated. The results suggest that BLACaMKII‐rACC mediates the mechanical allodynia and anxiety‐like behaviors in mice.

Our previous findings suggest EA alleviated anxiety‐like behaviors induced by neuropathic pain by regulating BLA. 13 In addition, EA alleviated anxiety‐like behaviors caused by chronic pain through rACC. 25 However, it is still unclear whether the analgesic and anxiolytic effects of EA are associated with the BLACaMKII‐rACC neural circuit. Therefore, in the SNI‐mCherry‐CNO‐EA group, we performed EA intervention on the SNI mice. In the SNI‐hM4D‐CNO‐EA group, we first administered CNO to inhibit bilateral BLACaMKII‐rACC in SNI mice, followed by EA intervention.

The results indicated that mechanical allodynia and anxiety‐like behaviors were more severe in the SNI‐hM4D‐CNO‐EA group compared to the SNI‐mCherry‐CNO‐EA group, suggesting that inhibition of the BLACaMKII‐rACC neural circuit antagonized the analgesic and anxiolytic‐like effects of EA. Thus, the analgesic and anxiolytic effects of EA can be inferred to be associated with the BLACaMKII‐rACC neural circuit.

5 CONCLUSIONS

In summary, we verified the analgesic and anxiety‐like effects of 2 Hz EA in SNI mice and may be associated with BLA CaMKII neuron activity. Besides, we have demonstrated a neural circuit mechanism in which BLACaMKII‐rACC plays a crucial role in mechanical allodynia and anxiety‐like behaviors. Additionally, BLACaMKII‐rACC may mediate the analgesic and anxiolytic effects of 2 Hz EA.

AUTHOR CONTRIBUTIONS

Yuerong Chen, Siyuan Tong, Yingling Xu, Yunyun Xu, Zonglin Wu, Xixiao Zhu, Xirui Wang, Chaoran Li, Chalian Lin, Xiaoyu Li, Chi Zhang, Yifang Wang: performed experiments and analyzed data. Yuanyuan Wu, Jianqiao Fang, Xiaomei Shao: design of experimental protocols, supervision and conceptualization. Yuerong Chen, Siyuan Tong: manuscript writing and editing. Yuanyuan Wu, Jianqiao Fang, Yunyun Xu, Yuerong Chen, Xiaoyu Li: funding acquisition.

FUNDING INFORMATION

This work was funded by the National Natural Science Foundation of China (Grant number: 82074541); Natural Science Foundation of Zhejiang Province (Grant number: LY19H270007, LY23H270009, LQ24H270003); Zhejiang Chinese Medical University (Grant number: 2021YKJ08, 2021YKJ09); and the National College Students’ Innovation and Entrepreneurship Training Program of China (Grant number: 202210344020).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflict of interest.

ACKNOWLEDGMENTS

The authors would like to thank the support of the National Natural Science Foundation of China (Grant number: 82074541); Natural Science Foundation of Zhejiang Province (Grant number: LY19H270007, LY23H270009, LQ24H270003); Zhejiang Chinese Medical University (Grant number: 2021YKJ08, 2021YKJ09); National College Students' Innovation and Entrepreneurship Training Program of China (Grant number: 202210344020).

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.
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REFERENCES

1 Cohen SP , Vase L , Hooten WM . Chronic pain: an update on burden, best practices, and new advances. Lancet. 2021;397 :2082‐2097.34062143
2 Yamauchi N , Sato K , Sato K , et al. Chronic pain‐induced neuronal plasticity in the bed nucleus of the stria terminalis causes maladaptive anxiety. Sci Adv. 2022;8 :eabj5586.35476439
3 Zhuo M . Neural mechanisms underlying anxiety‐chronic pain interactions. Trends Neurosci. 2016;39 :136‐145.26878750
4 Carvalho AF , Sharma MS , Brunoni AR , Vieta E , Fava GA . The safety, tolerability and risks associated with the use of newer generation antidepressant drugs: a critical review of the literature. Psychother Psychosom. 2016;85 :270‐288.27508501
5 Adamo D , Calabria E , Coppola N , Pecoraro G , Mignogna MD . Vortioxetine as a new frontier in the treatment of chronic neuropathic pain: a review and update. Ther Adv Psychopharmacol. 2021;11 :20451253211034320.34497709
6 Zirak N , Shafiee M , Soltani G , Mirzaei M , Sahebkar A . Hypericum perforatum in the treatment of psychiatric and neurodegenerative disorders: current evidence and potential mechanisms of action. J Cell Physiol. 2019;234 :8496‐8508.30461013
7 Sobieraj DM , Martinez BK , Hernandez AV , et al. Adverse effects of pharmacologic treatments of major depression in older adults. J Am Geriatr Soc. 2019;67 :1571‐1581.31140587
8 Jang J‐H , Song EM , do YH , et al. Acupuncture alleviates chronic pain and comorbid conditions in a mouse model of neuropathic pain: the involvement of DNA methylation in the prefrontal cortex. Pain. 2021;162 :514‐530.32796318
9 Zhang X‐H , Feng CC , Pei LJ , et al. Electroacupuncture attenuates neuropathic pain and comorbid negative behavior: the involvement of the dopamine system in the amygdala. Front Neurosci. 2021;15 :657507.34025342
10 Liu J , Li D , Huang J , et al. Glutamatergic neurons in the amygdala are involved in paclitaxel‐induced pain and anxiety. Front Psych. 2022;13 :869544.
11 Zeitler A , Kamoun N , Goyon S , et al. Favouring inhibitory synaptic drive mediated by GABA(a) receptors in the basolateral nucleus of the amygdala efficiently reduces pain symptoms in neuropathic mice. Eur J Neurosci. 2016;43 :1082‐1088.26913957
12 Matsuzawa‐Yanagida K , Narita M , Nakajima M , et al. Usefulness of antidepressants for improving the neuropathic pain‐like state and pain‐induced anxiety through actions at different brain sites. Neuropsychopharmacology. 2008;33 :1952‐1965.17957217
13 Wu M , Chen Y , Shen Z , et al. Electroacupuncture alleviates anxiety‐like behaviors induced by chronic neuropathic pain via regulating different dopamine receptors of the basolateral amygdala. Mol Neurobiol. 2022;59 :5299‐5311.35696012
14 Li J , Wang X , Wang H , et al. The BDNF‐TrkB signaling pathway in the rostral anterior cingulate cortex is involved in the development of pain aversion in rats with bone cancer via NR2B and ERK‐CREB signaling. Brain Res Bull. 2022;185 :18‐27.35398541
15 Zhang L , Wang G , Ma J , et al. Brain‐derived neurotrophic factor (BDNF) in the rostral anterior cingulate cortex (rACC) contributes to neuropathic spontaneous pain‐related aversion via NR2B receptors. Brain Res Bull. 2016;127 :56‐65.27575004
16 Mussio CA , Harte SE , Borszcz GS . Regional differences within the anterior cingulate cortex in the generation versus suppression of pain affect in rats. J Pain. 2020;21 :121‐134.31201992
17 Ma X , Yu W , Yao P’ , et al. Afferent and efferent projections of the rostral anterior cingulate cortex in young and middle‐aged mice. Front Aging Neurosci. 2022;14 :960868.36062147
18 Zhang Y , Meng X , Li A , et al. Acupuncture alleviates the affective dimension of pain in a rat model of inflammatory hyperalgesia. Neurochem Res. 2011;36 :2104‐2110.21695393
19 Zhou W , Jin Y , Meng Q , et al. A neural circuit for comorbid depressive symptoms in chronic pain. Nat Neurosci. 2019;22 :1649‐1658.31451801
20 Zhu X et al. Rostral anterior cingulate cortex‐ventrolateral periaqueductal gray circuit underlies Electroacupuncture to alleviate hyperalgesia but not anxiety‐like behaviors in mice with spared nerve injury. Front Neurosci. 2021;15 :757628.35095390
21 Shields SD , Eckert WA , Basbaum AI . Spared nerve injury model of neuropathic pain in the mouse: a behavioral and anatomic analysis. J Pain. 2003;4 :465‐470.14622667
22 Pentkowski NS , Rogge‐Obando KK , Donaldson TN , Bouquin SJ , Clark BJ . Anxiety and Alzheimer's disease: behavioral analysis and neural basis in rodent models of Alzheimer's‐related neuropathology. Neurosci Biobehav Rev. 2021;127 :647‐658.33979573
23 Lezak KR , Missig G , Carlezon WA . Behavioral methods to study anxiety in rodents. Dialogues Clin Neurosci. 2017;19 :181‐191.28867942
24 Zhou F , Jiang H , Kong N , et al. Electroacupuncture attenuated anxiety and depression‐like behavior via inhibition of hippocampal inflammatory response and metabolic disorders in TNBS‐induced IBD rats. Oxid Med Cell Longev. 2022;2022 :8295580.35087621
25 Wu Z , Shen Z , Xu Y , et al. A neural circuit associated with anxiety‐like behaviors induced by chronic inflammatory pain and the anxiolytic effects of electroacupuncture. CNS Neurosci Ther. 2023;30 :e14520.38018559
26 Li X , Zhu Y , Sun H , et al. Electroacupuncture inhibits pain memory and related anxiety‐like behaviors by blockading the GABAB receptor function in the Midcingulate cortex. Mol Neurobiol. 2023;60 :6613‐6626.37468738
27 Peirs C , Williams SPG , Zhao X , et al. Mechanical allodynia circuitry in the dorsal horn is defined by the nature of the injury. Neuron. 2021;109 :73‐90.e7.33181066
28 Huang J , Gadotti VM , Chen L , et al. A neuronal circuit for activating descending modulation of neuropathic pain. Nat Neurosci. 2019;22 :1659‐1668.31501573
29 Lu J‐S , Yang L , Chen J , et al. Basolateral amygdala astrocytes modulate diabetic neuropathic pain and may be a potential therapeutic target for koumine. Br J Pharmacol. 2023;180 :1408‐1428.36519959
30 Grossman N , Bono D , Dedic N , et al. Noninvasive deep brain stimulation via temporally interfering electric fields. Cell. 2017;169 :1029‐1041.e16.28575667
31 Ren W‐H , Guo JD , Cao H , et al. Is endogenous D‐serine in the rostral anterior cingulate cortex necessary for pain‐related negative affect? J Neurochem. 2006;96 :1636‐1647.16476080
32 Gard T , Holzel BK , Sack AT , et al. Pain attenuation through mindfulness is associated with decreased cognitive control and increased sensory processing in the brain. Cereb Cortex. 2012;22 :2692‐2702.22172578
33 Deng M , Zou W . Noncoding RNAs: novel targets for opioid tolerance. Curr Neuropharmacol. 2023;21 :1202‐1213.36453497
34 Feingold D , Weiser M , Rehm J , Lev‐Ran S . The association between cannabis use and anxiety disorders: results from a population‐based representative sample. Eur Neuropsychopharmacol. 2016;26 :493‐505.26775742
35 Venkatachalam AB , Levesque B , Achenbach JC , Pappas JJ , Ellis LD . Long and short duration exposures to the selective serotonin reuptake inhibitors (SSRIs) fluoxetine, paroxetine and sertraline at environmentally relevant concentrations Lead to adverse effects on zebrafish behaviour and reproduction. Toxics. 2023;11 :151.36851026
36 Marks DM et al. Serotonin‐norepinephrine reuptake inhibitors for pain control: premise and promise. Curr Neuropharmacol. 2009;7 :331‐336.20514212
37 Golder S , Medaglio D , O'Connor K , Hennessy S , Gross R , Gonzalez Hernandez G . Reasons for discontinuation or change of selective serotonin reuptake inhibitors in online drug reviews. JAMA Netw Open. 2023;6 :e2323746.37459097
38 Rothmore J . Antidepressant‐induced sexual dysfunction. Med J Aust. 2020;212 :329‐334.32172535
39 Simon G . Review: in older adults with acute major depression, SNRIs, but not SSRIs, increase adverse events vs placebo. Ann Intern Med. 2019;171 :JC39.31610552
40 Li Y , Liu X , Fu Q , et al. Electroacupuncture ameliorates depression‐like behaviors comorbid to chronic neuropathic pain via Tet1‐mediated restoration of adult neurogenesis. Stem Cells. 2023;41 :384‐399.36648299
41 Birch S , Lee MS , Alraek T , Kim T‐H . Evidence, safety and recommendations for when to use acupuncture for treating cancer related symptoms: a narrative review. Integr Med Res. 2019;8 :160‐166.31304088
42 Hullender Rubin LE , Smith CA , Schnyer RN , Tahir P , Pasch LA . Effect of acupuncture on IVF‐related anxiety: a systematic review and meta‐analysis. Reprod Biomed Online. 2022;45 :69‐80.35570176
43 Neugebauer V , Mazzitelli M , Cragg B , Ji G , Navratilova E , Porreca F . Amygdala, neuropeptides, and chronic pain‐related affective behaviors. Neuropharmacology. 2020;170 :108052.32188569
44 Luo Z‐Y , Huang L , Lin S , et al. Erbin in amygdala Parvalbumin‐positive neurons modulates anxiety‐like behaviors. Biol Psychiatry. 2020;87 :926‐936.31889536
45 Babaev O , Cruces‐Solis H , Piletti Chatain C , et al. IgSF9b regulates anxiety behaviors through effects on centromedial amygdala inhibitory synapses. Nat Commun. 2018;9 :5400.30573727
46 Savarese A , Lasek AW . Regulation of anxiety‐like behavior and Crhr1 expression in the basolateral amygdala by LMO3. Psychoneuroendocrinology. 2018;92 :13‐20.29609111
47 Duan K , Gu Q , Petralia RS , et al. Mitophagy in the basolateral amygdala mediates increased anxiety induced by aversive social experience. Neuron. 2021;109 :3793‐3809.e8.34614419
48 Sah P . Fear, anxiety, and the amygdala. Neuron. 2017;96 :1‐2.28957662
49 Helmstetter FJ . Stress‐induced hypoalgesia and defensive freezing are attenuated by application of diazepam to the amygdala. Pharmacol Biochem Behav. 1993;44 :433‐438.8446677
50 Zheng Z‐H , Tu JL , Li XH , et al. Neuroinflammation induces anxiety‐ and depressive‐like behavior by modulating neuronal plasticity in the basolateral amygdala. Brain Behav Immun. 2021;91 :505‐518.33161163
51 Juarez‐Salinas DL , Braz JM , Etlin A , Gee S , Sohal V , Basbaum AI . GABAergic cell transplants in the anterior cingulate cortex reduce neuropathic pain aversiveness. Brain. 2019;142 :2655‐2669.31321411
52 Szekely A , Silton RL , Heller W , Miller GA , Mohanty A . Differential functional connectivity of rostral anterior cingulate cortex during emotional interference. Soc Cogn Affect Neurosci. 2017;12 :476‐486.27998997
53 Xiao X , Yang Y , Zhang Y , Zhang XM , Zhao ZQ , Zhang YQ . Estrogen in the anterior cingulate cortex contributes to pain‐related aversion. Cereb Cortex. 2013;23 :2190‐2203.22784608
54 Sun J , Zhang C , Wang Y , et al. Electroacupuncture alleviates hyperalgesia and anxiety‐like behaviors in pain memory model rats through activation of GABAergic neurons and GABA receptor in the rostral anterior cingulate cortex. Mol Neurobiol. 2024;61 :6613‐6627.38329679
55 Xu Y , Zhu X , Chen Y , et al. Electroacupuncture alleviates mechanical allodynia and anxiety‐like behaviors induced by chronic neuropathic pain via regulating rostral anterior cingulate cortex‐dorsal raphe nucleus neural circuit. CNS Neurosci Ther. 2023;29 :4043‐4058.37401033
