Mechanisms of Electroacupuncture in Alleviating Stress-induced Irritable Bowel Syndrome in Rats: The Role of the Central Amygdala

Mechanisms of Electroacupuncture in Alleviating Stress-induced Irritable Bowel Syndrome in Rats: The Role of the Central Amygdala | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanisms of Electroacupuncture in Alleviating Stress-induced Irritable Bowel Syndrome in Rats: The Role of the Central Amygdala Rongxin Weng, Qinghui Wei, Zepeng Wang, Xinyu Li, Yuanyuan Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3876166/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Visceral hypersensitivity (VH) and anxiety are the main characteristics of irritable bowel syndrome (IBS) patients. Acupuncture has been demonstrated to be effective as a non-pharmacological approach for treating IBS. The central amygdala (CeA) is a key brain nucleus closely related to mediate integration of abdominal pain, gastrointestinal motility and anxiety. In the present study, we investigated whether and how the CeA mediate the protective effects of electroacupuncture (EA) against VH and anxiety in water avoidance stress (WAS)-induced IBS rats. Visceral sensitivity was assessed via abdominal withdrawal reflex and diarrhea index. Anxiety-related behavior was measured via open field test. The expression of c-Fos, GAD65, SYN1, PSD95 and BDNF in CeA were measured by using immunofluorescence or western blot. The chemogenetic method was used to activate or inhibit the selected target neurons. We found that EA attenuated WAS-induced gastrointestinal symptoms and anxiety-like behaviors with reduced expression of c-Fos, GAD65, SYN1, PSD95 and BDNF in the CeA. WAS-induced dysfunction was alleviated by inhibiting CeA neurons and the effects of EA treatment were counteracted by activating CeA neurons. Moreover, activating and inhibiting gamma-aminobutyric acidergic (GABAergic) neurons in the CeA can impede and enhance the effects of EA treatment, respectively. These data suggest that EA is effective to ameliorate stress-induced VH and anxiety via alleviating neuronal hyperactivity in the CeA, particularly targeting the hyperactivity of GABAergic neurons in the CeA, and suppressing enhanced synaptic plasticity in this region. Irritable bowel syndrome (IBS) Electroacupuncture (EA) Central amygdala (CeA) Visceral hypersensitivity (VH) Anxiety Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Irritable bowel syndrome (IBS) is a chronic gastrointestinal (GI) disorder that affects about 5–10% of the population of most western countries and China [ 1 ]. Based on the ROME criteria [ 2 ], IBS is defined by chronically recurring abdominal pain associated with altered bowel habits in the absence of histological damage within the gastrointestinal tract. An increasing number of studies have proven that IBS is a disorder of dysregulation of the brain-gut axis perturbed by various environmental factors. Many brain nuclei have been found to be involved in the central neuromodulation mechanism responsible for the development of IBS [ 3 – 6 ]. One of the key nuclei is the amygdala, especially the central nucleus of the amygdala (CeA), which is known to play a crucial role in the coding and modulation of pain and related emotions [ 7 ]. Many clinical trials have indicated that acupuncture is effective and safe for patients with IBS [ 8 – 10 ]. Positive effects and underlying mechanisms of acupuncture have also been preliminarily demonstrated in different animal models of IBS. Previous studies have shown that signals generated by acupuncture can modulate the central structures and interconnected neural circuits of multiple brain regions, including the medulla oblongata, thalamus, hypothalamus and cerebral cortex [ 11 – 14 ]. The analgesic process also involves the participation of various neurotransmitters, such as 5-hydroxytryptamine, glutamate and enkephalin [ 11 ]. What we concern is whether and how the CeA is involved in mediating the regulatory effects of acupuncture treatment of IBS. The CeA is striatum-like and the vast majority of CeA neurons are gamma-aminobutyric acidergic (GABAergic) exhibiting medium spiny-type morphology [ 15 – 17 ] In addition to participating in the formation of internal inhibitory circuits, these CeA neurons project widely to autonomic regions including the brainstem and forebrain areas that mediate not only anxiety-related behaviors but also enteric processes such as gastric emptying and colonic motility [ 18 – 19 ]. Neuroplasticity in the CeA is positively correlated with visceral pain behavior in animals [ 20 ]. However, to our knowledge, there is still no clear evidence and conclusions about the role of CeA in the mediating mechanism of acupuncture treatment for IBS, particularly based on experimental data. The current study was designed to investigate whether and how the CeA mediate the protective effects of electroacupuncture (EA) against visceral hypersensitivity (VH) and anxiety in stress-induced IBS rats. A 7-day chronic water avoidance stress (WAS) was used to induce diarrhea-predominant IBS (D-IBS) in Wistar rats. Visceral sensitivity was assessed via abdominal withdrawal reflex (AWR) and diarrhea index (DI). Anxiety-related behavior was assessed via open field test (OFT). The expression of c-Fos, GAD65, SYN1, PSD95 and BDNF in CeA were measured using immunofluorescence or western blot. To further determine the role of CeA neurons, the chemogenetic method was used to active or inhibit the selected target neurons in IBS rats. Materials and Methods Animals Male adult Wistar rats (220–280 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. All animals were kept individually under controlled temperature (20–24°C), humidity (50%-70%) and light conditions (lights on 6:00–18:00) and fed with food and water ad libitum for 1 week before the start of the experiment. All experimental procedures were performed in accordance with the National Institutes of Health’s ‘Guide for the Care and Use of Laboratory Animals’ and were approved by the Animal Experimental Ethics Committee of Shandong Normal University (reference no. AEECSDNU2020003). All efforts were made to minimize suffering and the number of animals used. Stress protocols and bowel function recording The chronic WAS protocol was modified according to the procedure previously described [ 21 ]. Briefly, a platform (10 cm length×8 cm width×8 cm height) was fixed in the center of an organic glass pool (45 cm length×25 cm width×25 cm height) filled with water at 25 ◦C to within 1 cm of the top of the platform. The rats were placed on the platform for a period of 1 h daily for 7 consecutive days. The frequency of defecation during the hour were counted. Sham rats were placed on the same platform without water in the pool. The diarrhea index (DI) during a 12 h period after the final WAS intervention of each rat was calculated by the loose stool rate × the average loose stool grade [ 22 ]. Loose stool rate was calculated by frequency of loose stool / total frequency of stool × 100%. Loose stool grade was evaluated according to the staining diameter of the stool on filter paper: Grade 1, diameter < 1 cm; Grade 2, diameter between1–1.9 cm; Grade 3, diameter between 2–2.9 cm; Grade 4, diameter ≥ 3 cm. Virus construction and infection Adeno-associated viruses (AAV9) were designed to achieve the chemogenetic manipulation strategy. Firstly, AAV9-hSyn-hM3D(Gq)-mCherry and AAV9-hSyn-hM4D(Gi)-mCherry were chosen to excite and inhibit neurons of CeA respectively. Secondly, AAV9-GAD65-cre combined with AAV9-hSyn-DIO-hM3D(Gq)-mCherry or AAV9-hSyn-DIO-hM4D(Gi)-mCherry were used to selectively excite or inhibit GABAergic neurons in CeA respectively. AAV9-hSyn-mCherry was selected as the control virus in control rats. All the viruses were acquired from Shandong Vigene Biosciences Co., Ltd. Rats were anesthetized with isoflurane (output concentration 2%, oxygen flow 500 mL/min). The temperature was maintained and monitored with a thermometer during the operation. A midline incision of the skull was made to fully expose the bregma and lambda areas. According to the atlas of Paxinos & Watson [ 23 ], the CeA (coordinates: 2.2 mm posterior to bregma, 4.0 mm lateral to the midline and 8.0 mm ventral from dura) was located for viral injections. The desired viral vectors (400 nL) were injected into the CeA at a rate of 40 nL/min. The needle was left in place for 8 min at the end of the infusion to prevent virus overflow. Clozapine N-oxide (CNO, 2 mg/kg) was daily injected intraperitoneally(i.p.) to activate or inhibit target neurons 14 days after virus infection. Control rats were injected i.p. with an equal amount of normal saline (NS). If the viral infection area checked by immunofluorescence exceeded the CeA region, it was not included in the statistics. EA treatment Sterilized stainless steel needles (0.25mm in diameter, 13 mm in length; Huatuo, Suzhou, China) were quickly inserted through the skin and into the underlying muscles to a depth of 5mm at ST25 or ST36 bilaterally. ST25 is located 5 mm lateral to the intersection between the upper 2/3 and the lower 1/3 in the line joining the xiphoid process and the upper edge of pubic symphysis [ 24 ]. ST36 is located 5 mm below head of fibula under knee joint, and 2 mm lateral to the anterior tubercle of the tibia in rats [ 25 ]. The needle handles were connected to a Han’s acupoint nerve stimulator (HANS-200, Jisheng Medical Science and Technology Co., Ltd., Nanjing, China) and stimulation (2/100 Hz frequency, 1 mA intensity, 0.5 ms pulse width) was applied for 30min after each stress-loading, based on our previous study [ 26 ]. Open field test The open field (OFT) test is used to assess WAS-induced anxiety-like behaviors. According to the previous reports [ 27 ], The OFT apparatus was designed as a square arena surrounded by high walls (100cm×100×50cm) made of Plexiglas, with the floor divided equally into 16 squares (25cm×25cm). After a pre-test acclimation of at least 30 min in the testing rooms, each rat was removed from its home cage and placed into the center of the arena and allowed to explore it freely for 5 min. The entire process was recorded by the video camera suspended 200 cm above the center of the arena. By analyzing the video file, central time (the time that the rat spent in the central 50×50 cm area of the open field), central entries (the total number that the rat entered into the center area) and defecations (the total number of fecal boli of the rat) were obtained. More central time and central entries and less defecations are viewed as low levels of anxiety [ 28 – 29 ]. After each test session, the open field box was carefully wiped with ethanol solution (70%) and allowed to dry completely to avoid the presence of any odor traces of the previously evaluated animal. Abdominal withdrawal reflex scores and pain threshold pressure Visceral hypersensitivity (VH) was measured by abdominal withdrawal reflex (AWR) scores and pain threshold pressure (PTP) as described previously [ 30 – 31 ]. Briefly, under anesthesia with isofluorane, a flexible latex balloon with a diameter of 5 cm attached to a polyethylene tube was inserted 6 cm into the descending colon and rectum via anus and secured by taping the tube to the rat’s tail. Rats were housed in small lucite cubicles and allowed to adapt for 1 hour. Graded colorectal distention (CRD) of 20, 40, 60, and 80 mmHg was performed by rapidly inflating the balloon to constant pressure using a sphygmomanometer for 20 s followed by 2 min rest. A blinded observer observed the animal response and give a score of either 0 (no response), 1 (slight head movement followed by immobility), 2 (contraction of abdominal muscles), 3 (lifting of abdomen) or 4 (body arching and lifting of pelvic structures). Similarly, PTP was measured with CRD increased step by step at intervals of 5mmHg. The PTP was defined as the minimal pressure of the CRD when the rat showed lifting of abdomen. AWR scores and PTP were measured on the day after the completion of the 7-day WAS procedure. Each rat was tested twice and the mean AWR score and PTP from the two repeated measurements was used for each rat in the following statistical analysis. Immunofluorescence detection c-Fos immunofluorescence was determined as previously reported [ 32 ]. Briefly, rats were perfused with 0.01 M phosphate-buffered saline (PBS, pH 7.4) followed by 150 mL freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (4°C). The brains were removed immediately, post-fixed for 3 hours, and dehydrated overnight in 20% sucrose in 0.1 M phosphate buffer saline (PBS) at 4°C. Series of frozen coronal sections containing the CeA were cut at 30 µm in a cryostat and then collected into 0.01 M PBS. Endogenous active enzymes were blocked with 2% goat serum in 0.01 M PBS containing 0.5% Triton X-100 for 30 min at room temperature. The sections were incubated overnight at 4°C with rabbit anti-c-Fos (1:500; Abcam Trading Co. Ltd. Shanghai, China), followed by incubation with biotinylated goat anti-rabbit IgG (Hangzhou Hua’an Biotechnology Co. Ltd. Hangzhou, China) for 1 hour at room temperature. Images of the expression of c-Fos-positive neurons were obtained by a fluorescence microscope (Olympus Biological Microscope BX43, Japan). The number of c-Fos immunoreactive (IR) neurons in the CeA per unit area (0.01 mm2) was counted by using Image pro-Plus6.0 professional image analysis software (Media Cybernetics Inc, MD, USA). Western blot Western blot was used to detect the protein expression levels of GAD65, SYN1, PSD-95 and BDNF in the CeA. Bilateral CeAs were collected according to the atlas [ 23 ] and the proteins were extracted. 40µg of Proteins extract were separated by SDS-PAGE and then were transferred onto PVDF membranes. Subsequently, the membrane was blocked with a closure solution (5% defatted milk powder and 0.1% Tween-20 contained in 0.01 M PBS) at 4°C for 1 h, followed by incubation with primary antibodies: rabbit anti-Beta (1:10000; R1207-1; Huabio), rabbit anti-SYN1 (1:5000; 20258-1-AP; Proteintech), rabbit anti-PSD95(1:2000; ET1602-20; Huabio), rabbit anti-BDNF(1:10000; ET1606-42; Huabio) and mouse anti-GAD65( 1:1000;GB12562;servicebio) respectively overnight at 4℃. Then, the membranes were incubated with the secondary anti-rabbit or anti-mouse IgG antibodies for 1 hour at room temperature. Enhanced chemiluminescence staining was used and the membrane was scanned by image analysis software. Quantity One was used to measure the optical density. The amount of protein was quantified by densitometry and normalized to β-actin, an internal standard. Statistical analysis Data were expressed as mean ± SD, and analysed by one-way analysis of variance (ANOVA) using SPSS V.29.0 statistical software (SPSS Inc., Chicago, IL, USA). The post-hoc test of least significant difference or Tambane’s T2 test (equal variance not assumed) was used to compare mean values between groups. P < 0.05 were considered statistically significant. Results EA attenuated WAS-induced VH and anxiety-like behaviors The timeline of this part of the experiment is shown in Fig.1A. All the results shown in Fig.1 and Fig.2 are from the 4 group: sham group, WAS group, WAS+ ST25 group and WAS+ ST36 group (n=7/group). As shown in Fig.1B, the AWR scores showed no significant difference among 4 groups when the distension pressure was 20mmHg ( P >0.05) and 80mmHg (scores in all groups =4±0.00). AWR score in WAS group was significantly higher than that of sham group when the distension pressure was 40mmHg ( P <0.001) and 60mmHg ( P <0.001), dedicating WAS induced obvious VH. Compared with the WAS group, EA treatment at both ST25 and ST36 significantly decreased AWR score when the distension pressure was 40mmHg ( P =0.019 & P =0.049 respectively). However, EA treatment did not decrease significantly AWR score when the distension pressure was 60mmHg (both P >0.05). There was no significant difference between AWR scores in the EA+ST25 and EA+ST 36 group (all P >0.05). Similarly, as shown in Fig.1C, PTP in WAS group was significantly Lower than that of sham group ( P <0.001), also dedicating WAS induced VH. Compared with the WAS group, EA treatment at both ST25 and ST36 significantly increased PTP (both P <0.001). As shown in Fig.1D, Defecation frequency (DF) during the 1 hour-stress in each day in sham group was significantly lower than that of WAS, WAS+ST25 and WAS+ST36 group (all P <0.001), which was consistent with the fact that stress can lead to an increase in animal defecation behavior. As shown in Fig.1E, compared with the WAS group, both EA treatments at ST25 and ST36 decreased DI significantly (both P <0.001). To explore the effects of WAS and EA treatment in anxiety, the OFT was carried in the last day. As shown in Fig.1F-H, both central time and central entries in WAS group were significantly decreased than those in sham group (both P <0.001) and defecations in WAS group were significantly increased than that in sham group ( P <0.001), dedicating obvious anxiety-like behaviors in WAS-induced IBS rats. Compared with the WAS group, both EA treatments at ST25 and ST36 significantly increased the central time (both P <0.001), central entries (P=0.0175 & =0.023) and deceased defecations (both P <0.001). Taken together, these results indicate that WAS-induced VH, diarrhea and anxiety-Like behaviors in rats can be alleviated by EA treatment. The effects of EA at ST 25 or ST 36 have no significant difference. EA suppressed WAS-induced expression of c-Fos, GAD65, SYN1, PSD95 and BDNF in the CeA Example images of c-Fos expression by immunofluorescence in the anterior, middle and posterior CeA of the 4 group were taken from brain sections 2.16~2.76 mm posterior to bregma (Fig.2A-B). As shown in Fig.2C, significantly more c-Fos IR neurons in the anterior, middle and posterior CeA were respectively observed in the WAS group than the sham group (all P <0.001). EA treatment at ST25 and ST36 significantly decreased the number of Fos IR neurons in the CeA compared with the WAS group (all P 0.05) (Fig.2C). The results of western blot analysis of CeA from the 4 group are shown in Fig.2D-G. The expression of GAD65, SYN1, PSD95 and BDNF of CeA was significantly increased by WAS compared with the sham group (all P <0.001). EA at both ST25 and ST 36 significantly inhibited GAD65 (both P <0.001), SYN1 (both P <0.001), PSD95 (both P <0.001) and BDNF ( P =0.001 & =0.028) expression in CeA compared with the untreated WAS group. The expression of GAD65, SYN1, PSD95 and BDNF showed no significant difference between the EA+ST25 and EA+ST 36 group. WAS-induced dysfunction was alleviated by inhibiting CeA neurons To verify the role of CeA in the development of stress-induced IBS, we used AAV9-hSyn-hM3D(Gq)-mCherry and AAV9-hSyn -hM4D(Gi)-mCherry respectively to activate and inhibit CeA neurons. The timeline of this part of the experiment is shown in Fig.3A. Immunofluorescence experiments were performed to validate the specificity of the viruses (Fig.3B). Compared with injecting AAV9-hSyn-mCherry, injecting AAV9-hSyn-hM3D(Gq)-mCherry increased c-Fos expression ( P <0.001) and injecting AAV9-hSyn-hM4D(Gi)-mCherry decreased c-Fos expression ( P <0.001) significantly (Fig.3C). The results in Fig.3D-I were from the 4 group: AAV9-hSyn-mCherry+NS (sham) group, AAV9-hSyn-mCherry+WAS+NS (WAS) group, AAV9-hSyn-hM3D(Gq)-mCherry+WAS+CNO (WAS+CeA activation) group and AAV9-hSyn-hM4D(Gi)-mCherry +WAS+CNO (WAS+CeA inhibition) group (n=7/group). As shown in Fig.3D-I, compared with WAS group, inhibiting CeA neurons significantly decreased AWR scores ( P =0.032) in response to CRD of 40mmHg, increased PTP ( P <0.001), decreased DI ( P <0.001), increased the central time and central entries ( P <0.001 & =0.0175) and decreased the defecations ( P <0.001). And all the data indicated no significant difference between the WAS group and the WAS+CeA activation group. These results demonstrate that inhibiting CeA neurons can effectively reverse WAS-induced VH, diarrhea and anxiety-like behaviors in rats. Activating CeA neurons blocked the effects of EA treatment This part was to investigate whether activating CeA neurons can block the effects of EA treatment. Because the effects of EA at ST 25 and ST 36 are similar in the first part of the experiment, only EA at ST 25 was observed in this part. The timeline of this part of the experiment is shown in Fig.4A. These results were from the 3 groups: AAV9-hSyn-mCherry+WAS+EA(WAS+EA) group, AAV9-hSyn-hM3D(Gq)-mCherry+WAS+CNO（WAS+ CeA activation）group and AAV9-hSyn-hM3D(Gq)-mCherry+WAS+CNO +EA（WAS+ CeA activation +EA）group (n=7/group) . As shown in Fig.4B-C, significantly more c-Fos IR neurons in the CeA were observed in the WAS+ CeA activation group and the WAS+ CeA activation+EA group respectively than WAS+EA group (both P <0.001). Compared with WAS+ CeA activation group, EA decreased the c-Fos expression in the CeA ( P =0.013). As shown in Fig.4D-I, compared with WAS+EA group, activating CeA neurons significantly increased AWR scores ( P =0.009) in response to CRD of 40mmHg, decreased PTP ( P =0.012), increased DI ( P =0.001), decreased the central time and central entries (both P <0.001) and increased the defecations ( P <0.001) in the WAS+ CeA activation+EA group. And all the data indicated no significant difference between the the WAS+ CeA activation group and the WAS+ CeA activation+EA group. These results demonstrate that activating CeA neurons can effectively block the effect of EA treatment on IBS rat. Activating or inhibiting the GABAergic neurons in the CeA respectively blocked or promoted the effects of EA treatment This part was to investigate the possible role of CeA GABAergic neurons in the mediating mechanism of EA intervention. The timeline of this part of the experiment is shown in Fig.5A. Immunofluorescence experiments were performed to validate the specificity of the viruses (Fig.5B). The results in Fig.5C-H were from the 3 groups: AAV9-hSyn-mCherry+WAS+EA(WAS+EA) group, AAV9-GAD65-cre+AAV9-hSyn-DIO-hM3D(Gq)-mCherry+WAS+CNO+EA (WAS+GABA activation+EA) group and AAV9-GAD65-cre+AAV9-hSyn-DIO-hM4D(Gi)-mCherry+WAS+CNO+EA (WAS+GABA inhibition +EA) group (n=7/group). compared with WAS+EA group, activating GABAergic neurons in the CeA significantly increased AWR scores ( P =0.013) in response to CRD of 40mmHg, decreased PTP ( P =0.0072), increased DI ( P <0.001), decreased the central time and central entries (both P <0.001) and increased the defecations ( P <0.001) in the WAS+GABA activation+EA group; and inhibiting GABAergic neurons in the CeA significantly decreased AWR scores ( P =0.041) in response to CRD of 40mmHg, increased PTP ( P =0.0012), decreased DI ( P =0.0409), increased the central time and central entries ( P <0.01=0.0371 & =0.0023, respectively) and decreased the defecations ( P =0.01) in the WAS+GABA inhibition+EA group. These findings suggest that activating or inhibiting GABAergic neurons in the CeA can impede and enhance the effects of EA treatment on IBS rats, respectively. Discussion In this study, we observed the intervention effects of EA on stress-induced IBS rats, and explored the mediating mechanisms involving the CeA. EA suppressed the neuronal overactivity and the enhancement of neuronal plasticity induced by stress, suggesting a crucial central mechanism against stress-induced VH and anxiety. The CeA and its GABAergic neurons may serve as important target nuclei and target neurons for EA intervention. Our study, for the first time, reveals the regulatory role of the CeA in mediating the effects of acupuncture on gastrointestinal symptoms and anxiety in IBS rats. In present study, A 7-day chronic WAS induced notably VH and anxiety in rats with increased c-Fos IR neurons in CeA, consistent with previous brain imaging studies in patients with IBS[ 19 , 33 ] and immunohistochemical experiment in mice[ 34 ] Our study found that EA intervention not only alleviated WAS-induced symptoms including abdominal pain, diarrhea and anxiety-like behavior but also suppressed the heightened expression of c-Fos in the CeA induced by WAS, suggesting a potential mediating role of the CeA. To confirm this point, we use chemogenetic method to induce the activation or inhibition of CeA neurons respectively. And we found that WAS-induced dysfunction was alleviated by inhibition of CeA neurons and that the effects of EA treatment was blocked by activating CeA neurons. These data suggest that EA is effective to ameliorate stress-induced VH and anxiety via inhibiting neuronal hyperactivity in the CeA. Neurontrophin, especially brain derived neurotrophic factor (BDNF) expression is also regulated by stress- and activity-dependent factor and induce the change of neuronal plasticity, so it is considered a potential biomarker of many stress-related diseases including anxiety and depression [ 35 ]. The experiment on the effect of BDNF on synaptic morphological differentiation in hippocampus proves that in the presence of BDNF, the primary dendrites and dendritic spines in the culture medium increase significantly [ 36 ]. Interestingly, the trends of stress-induced change of BDNF expression in different brain area are not consistent. For example, the BDNF expression is downregulated in the hippocampus but upregulated in the amygdala in stress response [ 37 – 38 ]. Increased BDNF expression can enhance synaptic plasticity in the CeA, both of which contribute to stress-induced VH [ 39 ]. In the present study, EA intervention suppressed the increasing expression of BDNF, SYN1 and PSD95 in the CeA induced by WAS. Therefore, we postulate that modulating synaptic plasticity may be a key mechanism underlying the beneficial effects of EA in ameliorating visceral pain. Notably, recent experiments have demonstrated that synaptic plasticity within the CeA is modulated by microglia after exposure to chronic stress [ 39 , 40 ]. Therefore, in further research, exploring whether microglia within the CeA could be considered as potential targets for acupuncture to intervene in IBS deserves attention. In the current study, we observed that WAS induced significant higher expression of GAD 65 in CeA, indicating increased GABA synthesis and activated GABAergic neurons. EA decreased the level of GAD65 expression in CeA, suggesting the possible mediating role of GABAergic neurons in the CeA. To confirm this point, we use chemogenetic method again to induce the activation and inhibition of GABAergic neurons in the CeA respectively. And we found activating and inhibiting GABAergic neurons in the CeA can, respectively, impede and enhance the effects of EA treatment on IBS rats. These data suggest that EA is effective to ameliorate stress-induced VH and anxiety via inhibiting the hyperactivity of GABAergic neurons in the CeA. However, GABAergic neurons in CeA are involved in several different inhibitory circuits or pathways inside or outside amygdala [ 19 ], and can act on the GABAA, GABAB and GABAC receptors [ 41 ]. Further investigation is needed to determine which specific inhibitory pathways are affected by EA and the mechanisms through which EA exerts its effects. Additionally, it is crucial to explore whether a specific GABA receptor is involved in mediating the regulatory effects of EA. In summary, our results demonstrated that inhibiting the excessive activity of neurons in the CeA, especially the excessive activity of GABAergic neurons, and suppressing enhanced synaptic plasticity in the CeA are potential mechanisms for the positive effects of EA in the treatment of IBS. Declarations Funding This work was supported by the Natural Science Foundation of Shandong Province (Grant numbers: ZR2021MH199, ZR2020MC075, ZR2021MC142 ) and the National Natural Science Foundation of China (32170496) Competing Interests The authors declare no competing interests. Author Contributions Rongxin Weng, Qinghui Wei, Zepeng Wang and Xinyu Li performed the experiments, analyzed the data, and contributed to study design and manuscript writing. Yuanyuan Li, Xuehan Sun, Yuxue Wang and Xiusong Wang performed the behavioral studies and contributed to data analyzing. Feng He and Haiji Sun supervised the study, analyzed data and wrote the manuscript. Data Availability Data supporting the results of this research can be obtained from the corresponding author upon reasonable request. Ethics approval This study was approved by the Animal Experimental Ethics Committee of Shandong Normal University (reference no. AEECSDNU2020003). Consent to Participate Not applicable. Consent to publish Not applicable. References Lovell RM, Ford AC (2012) Global prevalence of and risk factors for irritable bowel syndrome: a meta-analysis. Clin Gastroenterol Hepatol 10(7):712-721. https://doi.org/10.1016/j.cgh.2012.02.029 Drossman DA (2016) Functional gastrointestinal disorders: history, pathophysiology, clinical features, and Rome IV. Gastroenterology 150(6): 1262-1279.https://doi.org/10.1053/j.gastro.2016.02.032 Bai Y, Chen YB, Qiu XT, Chen YB, Ma LT, Li YQ et al (2019) Nucleus tractus solitarius mediates hyperalgesia induced by chronic pancreatitis in rats. 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J Comp Neurol 208(4):401-418.https://doi.org/10.1002/cne.902080409 Swanson LW, Petrovich GD (1998) What is the amygdala? Trends Neurosci 21(8):323-331. https://doi.org/10.1016/s0166-2236(98)01265-x Ehrlich I, Humeau Y, Grenier F, Ciocchi S, Herry C, Luthi A (2009) Amygdala inhibitory circuits and the control of fear memory. Neuron 62(6):757-771.https://doi.org/10.1016/j.neuron.2009.05.026 Mcdonald AJ, Mascagni F, Guo L (1996) Projections of the medial and lateral prefrontal cortices to the amygdala: a phaseolus vulgaris leucoagglutinin study in the rat. Neuroscience 71(1):55-75. https://doi.org/10.1016/0306-4522(95)00417-3 Ciocchi S, Herry C, Grenier F, Wolff SB, Letzkus JJ, Vlachos I et al (2010) Encoding of conditioned fear in central amygdala inhibitory circuits. Nature 468(7321):277-282.https://doi.org/10.1038/nature09559 Ji G, Li Z, Neugebauer V (2015) Reactive oxygen species mediate visceral pain-related amygdala plasticity and behaviors. Pain 156(5):825-836. https://doi.org/10.1097/j.pain.0000000000000120 Bradesi S, Schwetz I, Ennes HS, Lamy C, Ohning G, Fanselow M et al (2005) Repeated exposure to water avoidance stress in rats: a new model for sustained visceral hyperalgesia. Am J Physiol Gastrointest Liver Physiol 289(1): G42-G53.https://doi.org/10.1152/ajpgi.00500.2004 Xiao L, Cui T, Liu S, Chen B, Wang Y, Yang T et al (2019) Vitamin A supplementation improves the intestinal mucosal barrier and facilitates the expression of tight junction proteins in rats with diarrhea. Nutrition 57:97-108.https://doi.org/10.1016/j.nut.2018.06.007 Paxinos G, Watson C (2006) The rat brain in stereotaxic coordinates: hard cover edition. Elsevier Xu T, Yu Z, Liu Y, Lu M, Gong M, Li Q et al (2022) Hypoglycemic effect of electroacupuncture at ST25 through neural regulation of the pancreatic intrinsic nervous system. Mol Neurobiol 59(1):703-716.https://doi.org/10.1007/s12035-021-02609-1 Zhang S, Liu Y, Li S, Ye F, Yin J (2023) Autonomic and cytokine mechanisms of acute electroacupuncture in a rodent model of functional dyspepsia. Neurogastroenterology & Motility: e14702. https://doi.org/10.1111/nmo.14702 He F, Wang M, Geng X, Ai H (2018) Effect of electroacupuncture on the activity of corticotrophin-releasing hormone neurons in the hypothalamus and amygdala in rats exposed to restraint water-immersion stress. Acupunct Med 36(6):394-400.https://doi.org/10.1136/acupmed-2017-011450 Dandi E, Kalamari A, Touloumi O, Lagoudaki R, Nousiopoulou E, Simeonidou C et al (2018) Beneficial effects of environmental enrichment on behavior, stress reactivity and synaptophysin / BDNF expression in hippocampus following early life stress. Int J Dev Neurosci 67:19-32. https://doi.org/10.1016/j.ijdevneu.2018.03.003 Fulk LJ, Stock HS, Lynn A, Marshall J, Wilson MA, Hand GA (2004) Chronic physical exercise reduces anxiety-like behavior in rats. Int J Sports Med 25(1):78-82.https://doi.org/10.1055/s-2003-45235 Sarkar D (2020) A review of behavioral tests to evaluate different types of anxiety and anti-anxiety effects. Clin Psychopharmacol Neurosci 18(3):341-351.https://doi.org/10.9758/cpn.2020.18.3.341 Al-Chaer ED, Kawasaki M, Pasricha PJ (2000) A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development. Gastroenterology 119(5):1276-1285. https://doi.org/10.1053/gast.2000.19576 Yang JP, Yao M, Jiang XH, Wang LN (2006) Establishment of model of visceral pain due to colorectal distension and its behavioral assessment in rats. World J Gastroenterol 12(17):2781-2784. https://doi.org/10.3748/wjg.v12.i17.2781 Li LH, Ling DD, Lin H, Wang ZC, Sun ZR, Zhang YQ et al (2023) Ovariectomy induces hyperalgesia accompanied by upregulated estrogen receptor alpha and protein kinase B in the rat spinal cord. Physiol Behav 271:114342.https://doi.org/10.1016/j.physbeh.2023.114342 Naliboff BD, Derbyshire SW, Munakata J, Berman S, Mandelkern M, Chang L et al (2001) Cerebral activation in patients with irritable bowel syndrome and control subjects during rectosigmoid stimulation. Psychosom Med 63(3):365-375. https://doi.org/10.1097/00006842-200105000-00006 Reichmann F, Painsipp E, Holzer P (2013) Environmental enrichment and gut inflammation modify stress-induced c-Fos expression in the mouse corticolimbic system. PLoS One 8(1):e54811. https://doi.org/10.1371/journal.pone.0054811 Suliman S, Hemmings SMJ, Seedat S (2013) Brain-derived neurotrophic factor (BDNF) protein levels in anxiety disorders: systematic review and meta-regression analysis. S Afr J Psychiatr 19(3):124. https://doi.org/10.3389/fnint.2013.00055 Ji YY, Pang PT, Feng LY, Lu B (2005) Cyclic AMP controls BDNF-induced TrkB phosphorylation and dendritic spine formation in mature hippocampal neurons. Nat Neurosci 8(2):164-172. https://doi.org/10.1038/nn1381 Lakshminarasimhan, H. and S. Chattarji (2012). "Stress leads to contrasting effects on the levels of brain derived neurotrophic factor in the hippocampus and amygdala." PloS one 7(1): e30481. https://doi.org/10.1371/journal.pone.0030481 Chang, S.-H., Y. H. Yu, et al. (2021). "BDNF protein and BDNF mRNA expression of the medial prefrontal cortex, amygdala, and hippocampus during situational reminder in the PTSD animal model." Behavioural neurology 2021: 1-13. https://doi.org/10.1155/2021/6657716 Yuan T, Manohar K, Latorre R, Orock A, Meerveld BG (2020) Inhibition of microglial activation in the amygdala reverses stress-induced abdominal pain in the male rat. Cell Mol Gastroenterol Hepatol 10(3):527-543.https://doi.org/10.1016/j.jcmgh.2020.04.020 Yuan T, Orock A, Greenwood-Van MB (2021) Amygdala microglia modify neuronal plasticity via complement C1q/C3-CR3 signaling and contribute to visceral pain in a rat model. Am J Physiol Gastrointest Liver Physiol 320(6):G1081-G1092.https://doi.org/10.1152/ajpgi.00123.2021 Choi SI, Kim N, Nam RH, Jang JY, Kim EH, Ha S et al (2023) The protective effect of roseburia faecis against repeated water avoidance stress-induced irritable bowel syndrome in a wister rat model. J Cancer Prev 28(3):93-105.https://doi.org/10.15430/JCP.2023.28.3.93 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3876166","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":269439260,"identity":"1068627d-2cfb-4469-b065-733948a1e139","order_by":0,"name":"Rongxin Weng","email":"","orcid":"","institution":"Shandong Normal University","correspondingAuthor":false,"prefix":"","firstName":"Rongxin","middleName":"","lastName":"Weng","suffix":""},{"id":269439261,"identity":"7afdd1a3-297f-4c0a-adfc-da77996829a4","order_by":1,"name":"Qinghui Wei","email":"","orcid":"","institution":"Shandong Normal University","correspondingAuthor":false,"prefix":"","firstName":"Qinghui","middleName":"","lastName":"Wei","suffix":""},{"id":269439262,"identity":"a689e746-fea0-4c57-9497-87117e597deb","order_by":2,"name":"Zepeng Wang","email":"","orcid":"","institution":"Shandong Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zepeng","middleName":"","lastName":"Wang","suffix":""},{"id":269439267,"identity":"99738b5e-e938-4acf-9081-af0085a47dff","order_by":3,"name":"Xinyu Li","email":"","orcid":"","institution":"Shandong Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Li","suffix":""},{"id":269439269,"identity":"516801d7-a5c2-405f-8137-81f9f03288bd","order_by":4,"name":"Yuanyuan Li","email":"","orcid":"","institution":"Shandong Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Li","suffix":""},{"id":269439271,"identity":"82769d8e-36e5-48ea-a3bc-8721b7fe6b9a","order_by":5,"name":"Xuehan Sun","email":"","orcid":"","institution":"Shandong Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xuehan","middleName":"","lastName":"Sun","suffix":""},{"id":269439272,"identity":"60a4fe4e-85dc-4bed-bacc-62f3c07cc68c","order_by":6,"name":"Yuxue Wang","email":"","orcid":"","institution":"Shandong Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yuxue","middleName":"","lastName":"Wang","suffix":""},{"id":269439274,"identity":"1981b02e-9ba4-406a-b58f-6d0801c4b30c","order_by":7,"name":"Xiusong Wang","email":"","orcid":"","institution":"Shandong Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiusong","middleName":"","lastName":"Wang","suffix":""},{"id":269439275,"identity":"9833183d-c324-41a5-bee0-75f9905f79b3","order_by":8,"name":"Haiji Sun","email":"","orcid":"","institution":"Shandong Normal University","correspondingAuthor":false,"prefix":"","firstName":"Haiji","middleName":"","lastName":"Sun","suffix":""},{"id":269439277,"identity":"225a792a-31e9-48d9-b5b7-783701bf318a","order_by":9,"name":"Feng He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBAC9gYGBmYIq7Hx4QditPAcgGnhOdxsLEGaFon0NgEeorSw9x5+XVBx2N5c8mEbgwSDnZxuAyEtPOfSrGecOZy4c3Zi24MChmRjswMEtNhL5JgZ87YdTjC4ndhuIMFwIHEbIS088m+AWv4dtje4ebBNgocoLRI8xo95Gw4zbrjBSKwWnhwzZp5j6YkbziQCA9mACL/wsJ8x/sxTY21vcPz4w4cfKuzkCGoBAjZgBDZD2QaElYMAMzCZ1BGndBSMglEwCkYmAAChNUHlBf1A7wAAAABJRU5ErkJggg==","orcid":"","institution":"Shandong Normal University","correspondingAuthor":true,"prefix":"","firstName":"Feng","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2024-01-18 15:14:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3876166/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3876166/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50388466,"identity":"6021f384-d573-40db-a9d2-65428d10e330","added_by":"auto","created_at":"2024-01-30 18:08:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2596893,"visible":true,"origin":"","legend":"\u003cp\u003eEA attenuated WAS-induced visceral hypersensitivity (VH) and anxiety-like behaviors.\u003cstrong\u003e A \u003c/strong\u003eThe timeline of this part of the experiment. \u003cstrong\u003eB-E \u003c/strong\u003eComparisons of the abdominal withdrawal reflex (AWR) scores, pain threshold pressure(PTP), Defecation frequency(DF) during the 1 hour-stress in each day and diarrhea index（DI）in the last day between groups. \u003cstrong\u003eF-H\u003c/strong\u003e Comparisons of the central time, central entries and defecations in open field test (OFT) between groups. Data represent the means ± SD (n=7 per group). ** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 (compared with the sham group), # \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 and ## \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 (compared with the WAS group). WAS: water avoidance stress; AWR: abdominal withdrawal reflex; OFT: open field test; DI: diarrhea index; PTP: pain threshold pressure.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3876166/v1/991562a22f20a88698afb565.jpg"},{"id":50388870,"identity":"8fcd7644-7c42-4a58-9f30-9b919d145636","added_by":"auto","created_at":"2024-01-30 18:16:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3934686,"visible":true,"origin":"","legend":"\u003cp\u003eEA suppressed WAS-induced expression of Fos, GAD65, SYN1, PSD95 and BDNF expression in CeA.\u003cstrong\u003e A \u003c/strong\u003eReference planes (from 2.16mm to 2.76mm posterior from bregma) of the CEA. \u003cstrong\u003eB\u003c/strong\u003e Representative immunofluorescence images of c-Fos expression in the anterior, middle and posterior CEA of sham group, WAS group, WAS+ST25 group and WAS+ST36 group (scale bars: 100μm). \u003cstrong\u003eC\u003c/strong\u003eComparisons of the c-Fos expression between groups. Data represent the means ± SD (n=7 per group). ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 (compared with the sham group), # \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 and ## \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 (compared with the WAS group). \u003cstrong\u003eD-G\u003c/strong\u003e Representative Western blots image (upper part) and quantitative analysis (lower part) showing the expression of GAD65, SYN1, PSD95 and BDNF. Data represent the means ± SD (n=7 per group). ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 (compared with the sham group), # \u003cem\u003eP\u003c/em\u003e \u0026lt;0.05 and ## \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 (compared with the WAS group). CeA: central amygdala; aCeA: anterior CeA; mCeA: medium CeA; pCeA: posterior CeA; WAS: water avoidance stress.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3876166/v1/c3a27d58c707f7200ddd4450.jpg"},{"id":50388461,"identity":"61ddd538-a302-4c52-aefe-0092ec89c236","added_by":"auto","created_at":"2024-01-30 18:08:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3553444,"visible":true,"origin":"","legend":"\u003cp\u003eWAS-induced dysfunction was alleviated by Inhibiting CeA neurons. \u003cstrong\u003eA \u003c/strong\u003eThe timeline of this part of the experiment. \u003cstrong\u003eB \u003c/strong\u003eTypical immunofluorescence images of mCherry (red) and c-Fos (green) expression in the CeA (scale bars: 100μm). \u003cstrong\u003eC \u003c/strong\u003eA significant higher percent colocalization of c-Fos and mCherry in the hM3D group and a significant lower percent colocalization of c-Fos and mCherry in the hM4D group compared with mCherry group (** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, n=3 rats/group) \u003cstrong\u003eD-F \u003c/strong\u003eComparisons of the AWR scores, PTP and DI between groups (n=7 per group). \u003cstrong\u003eG-I\u003c/strong\u003eComparisons of the central time, central entries and defecations in OFT between groups. Data represent the means ± SD (n=7 per group). ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 (compared with the sham group), # \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 and ## \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 (compared with the WAS group). WAS: water avoidance stress; AWR: abdominal withdrawal reflex; OFT: open field test; DI: diarrhea index; PTP: pain threshold pressure.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3876166/v1/e0d70c410557aa6700e8ee51.jpg"},{"id":50388465,"identity":"a273dbb0-6641-4164-a7e2-1e2b4fa545b9","added_by":"auto","created_at":"2024-01-30 18:08:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3178327,"visible":true,"origin":"","legend":"\u003cp\u003eActivating CeA neurons blocked the effects of EA treatment. \u003cstrong\u003eA \u003c/strong\u003eThe timeline of this part of the experiment. \u003cstrong\u003eB \u003c/strong\u003eTypical immunofluorescence images of c-Fos (green) expression in each group(scale bars: 100μm). \u003cstrong\u003eC-F \u003c/strong\u003eComparisons of the c-Fos IR neurons, AWR scores, PTP and DI between groups. \u003cstrong\u003eG-I\u003c/strong\u003e Comparisons of the central time, central entries and defecations in OFT between groups. Data represent the means ± SD (n=7 per group). * \u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 (compared with the WAS+EA group). # \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 (compared with the WAS+CEA activation group) WAS: water avoidance stress, EA: electroacupuncture, CeA: central amygdala; AWR: abdominal withdrawal reflex; OFT: open field test; PTP: pain threshold pressure; DI: diarrhea index.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3876166/v1/ab1fff9bc183ea8a213f25d5.jpg"},{"id":50388463,"identity":"1e18ca08-39b9-4021-b066-0dd0c8680383","added_by":"auto","created_at":"2024-01-30 18:08:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2812956,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivating or inhibiting the GABAergic neurons in the CeA respectively blocked or promoted the effects of EA treatment\u003c/strong\u003e. \u003cstrong\u003eA \u003c/strong\u003eThe timeline of this part of the experiment. \u003cstrong\u003eB \u003c/strong\u003eTypical immunofluorescence images of mCherry (red) expression in the CeA (scale bars: 200μm) \u003cstrong\u003eC-E \u003c/strong\u003eComparisons of the AWR scores, PTP and DI between groups. \u003cstrong\u003eF-H\u003c/strong\u003e Comparisons of the central time, central entries and defecations in OFT between groups. Data represent the means ± SD (n=7 per group). * \u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 (compared with the WAS +EA group).WAS: water avoidance stress; CeA: central amygdala; OPT: optic tract; AWR: abdominal withdrawal reflex; PTP: pain threshold pressure; DI: diarrhea index; OFT: open field test.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3876166/v1/8802ef27777b5b1f94158148.jpg"},{"id":77289810,"identity":"b980ce77-b189-44f5-80a9-9c0a5d3411fa","added_by":"auto","created_at":"2025-02-27 06:11:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16866657,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3876166/v1/c1bcc593-fa2d-42a1-8245-05d37c34bb1a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanisms of Electroacupuncture in Alleviating Stress-induced Irritable Bowel Syndrome in Rats: The Role of the Central Amygdala","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIrritable bowel syndrome (IBS) is a chronic gastrointestinal (GI) disorder that affects about 5\u0026ndash;10% of the population of most western countries and China [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Based on the ROME criteria [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], IBS is defined by chronically recurring abdominal pain associated with altered bowel habits in the absence of histological damage within the gastrointestinal tract. An increasing number of studies have proven that IBS is a disorder of dysregulation of the brain-gut axis perturbed by various environmental factors. Many brain nuclei have been found to be involved in the central neuromodulation mechanism responsible for the development of IBS [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. One of the key nuclei is the amygdala, especially the central nucleus of the amygdala (CeA), which is known to play a crucial role in the coding and modulation of pain and related emotions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany clinical trials have indicated that acupuncture is effective and safe for patients with IBS [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Positive effects and underlying mechanisms of acupuncture have also been preliminarily demonstrated in different animal models of IBS. Previous studies have shown that signals generated by acupuncture can modulate the central structures and interconnected neural circuits of multiple brain regions, including the medulla oblongata, thalamus, hypothalamus and cerebral cortex [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The analgesic process also involves the participation of various neurotransmitters, such as 5-hydroxytryptamine, glutamate and enkephalin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhat we concern is whether and how the CeA is involved in mediating the regulatory effects of acupuncture treatment of IBS. The CeA is striatum-like and the vast majority of CeA neurons are gamma-aminobutyric acidergic (GABAergic) exhibiting medium spiny-type morphology [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] In addition to participating in the formation of internal inhibitory circuits, these CeA neurons project widely to autonomic regions including the brainstem and forebrain areas that mediate not only anxiety-related behaviors but also enteric processes such as gastric emptying and colonic motility [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Neuroplasticity in the CeA is positively correlated with visceral pain behavior in animals [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, to our knowledge, there is still no clear evidence and conclusions about the role of CeA in the mediating mechanism of acupuncture treatment for IBS, particularly based on experimental data.\u003c/p\u003e \u003cp\u003eThe current study was designed to investigate whether and how the CeA mediate the protective effects of electroacupuncture (EA) against visceral hypersensitivity (VH) and anxiety in stress-induced IBS rats. A 7-day chronic water avoidance stress (WAS) was used to induce diarrhea-predominant IBS (D-IBS) in Wistar rats. Visceral sensitivity was assessed via abdominal withdrawal reflex (AWR) and diarrhea index (DI). Anxiety-related behavior was assessed via open field test (OFT). The expression of c-Fos, GAD65, SYN1, PSD95 and BDNF in CeA were measured using immunofluorescence or western blot. To further determine the role of CeA neurons, the chemogenetic method was used to active or inhibit the selected target neurons in IBS rats.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale adult Wistar rats (220\u0026ndash;280 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. All animals were kept individually under controlled temperature (20\u0026ndash;24\u0026deg;C), humidity (50%-70%) and light conditions (lights on 6:00\u0026ndash;18:00) and fed with food and water ad libitum for 1 week before the start of the experiment. All experimental procedures were performed in accordance with the National Institutes of Health\u0026rsquo;s \u0026lsquo;Guide for the Care and Use of Laboratory Animals\u0026rsquo; and were approved by the Animal Experimental Ethics Committee of Shandong Normal University (reference no. AEECSDNU2020003). All efforts were made to minimize suffering and the number of animals used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStress protocols and bowel function recording\u003c/h2\u003e \u003cp\u003eThe chronic WAS protocol was modified according to the procedure previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Briefly, a platform (10 cm length\u0026times;8 cm width\u0026times;8 cm height) was fixed in the center of an organic glass pool (45 cm length\u0026times;25 cm width\u0026times;25 cm height) filled with water at 25 ◦C to within 1 cm of the top of the platform. The rats were placed on the platform for a period of 1 h daily for 7 consecutive days. The frequency of defecation during the hour were counted. Sham rats were placed on the same platform without water in the pool. The diarrhea index (DI) during a 12 h period after the final WAS intervention of each rat was calculated by the loose stool rate \u0026times; the average loose stool grade [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Loose stool rate was calculated by frequency of loose stool / total frequency of stool \u0026times; 100%. Loose stool grade was evaluated according to the staining diameter of the stool on filter paper: Grade 1, diameter\u0026thinsp;\u0026lt;\u0026thinsp;1 cm; Grade 2, diameter between1\u0026ndash;1.9 cm; Grade 3, diameter between 2\u0026ndash;2.9 cm; Grade 4, diameter\u0026thinsp;\u0026ge;\u0026thinsp;3 cm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eVirus construction and infection\u003c/h2\u003e \u003cp\u003eAdeno-associated viruses (AAV9) were designed to achieve the chemogenetic manipulation strategy. Firstly, AAV9-hSyn-hM3D(Gq)-mCherry and AAV9-hSyn-hM4D(Gi)-mCherry were chosen to excite and inhibit neurons of CeA respectively. Secondly, AAV9-GAD65-cre combined with AAV9-hSyn-DIO-hM3D(Gq)-mCherry or AAV9-hSyn-DIO-hM4D(Gi)-mCherry were used to selectively excite or inhibit GABAergic neurons in CeA respectively. AAV9-hSyn-mCherry was selected as the control virus in control rats. All the viruses were acquired from Shandong Vigene Biosciences Co., Ltd.\u003c/p\u003e \u003cp\u003eRats were anesthetized with isoflurane (output concentration 2%, oxygen flow 500 mL/min). The temperature was maintained and monitored with a thermometer during the operation. A midline incision of the skull was made to fully expose the bregma and lambda areas. According to the atlas of Paxinos \u0026amp; Watson [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the CeA (coordinates: 2.2 mm posterior to bregma, 4.0 mm lateral to the midline and 8.0 mm ventral from dura) was located for viral injections. The desired viral vectors (400 nL) were injected into the CeA at a rate of 40 nL/min. The needle was left in place for 8 min at the end of the infusion to prevent virus overflow. Clozapine N-oxide (CNO, 2 mg/kg) was daily injected intraperitoneally(i.p.) to activate or inhibit target neurons 14 days after virus infection. Control rats were injected i.p. with an equal amount of normal saline (NS). If the viral infection area checked by immunofluorescence exceeded the CeA region, it was not included in the statistics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEA treatment\u003c/h2\u003e \u003cp\u003eSterilized stainless steel needles (0.25mm in diameter, 13 mm in length; Huatuo, Suzhou, China) were quickly inserted through the skin and into the underlying muscles to a depth of 5mm at ST25 or ST36 bilaterally. ST25 is located 5 mm lateral to the intersection between the upper 2/3 and the lower 1/3 in the line joining the xiphoid process and the upper edge of pubic symphysis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. ST36 is located 5 mm below head of fibula under knee joint, and 2 mm lateral to the anterior tubercle of the tibia in rats [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The needle handles were connected to a Han\u0026rsquo;s acupoint nerve stimulator (HANS-200, Jisheng Medical Science and Technology Co., Ltd., Nanjing, China) and stimulation (2/100 Hz frequency, 1 mA intensity, 0.5 ms pulse width) was applied for 30min after each stress-loading, based on our previous study [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eOpen field test\u003c/h2\u003e \u003cp\u003eThe open field (OFT) test is used to assess WAS-induced anxiety-like behaviors. According to the previous reports [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], The OFT apparatus was designed as a square arena surrounded by high walls (100cm\u0026times;100\u0026times;50cm) made of Plexiglas, with the floor divided equally into 16 squares (25cm\u0026times;25cm). After a pre-test acclimation of at least 30 min in the testing rooms, each rat was removed from its home cage and placed into the center of the arena and allowed to explore it freely for 5 min. The entire process was recorded by the video camera suspended 200 cm above the center of the arena. By analyzing the video file, central time (the time that the rat spent in the central 50\u0026times;50 cm area of the open field), central entries (the total number that the rat entered into the center area) and defecations (the total number of fecal boli of the rat) were obtained. More central time and central entries and less defecations are viewed as low levels of anxiety [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. After each test session, the open field box was carefully wiped with ethanol solution (70%) and allowed to dry completely to avoid the presence of any odor traces of the previously evaluated animal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAbdominal withdrawal reflex scores and pain threshold pressure\u003c/h2\u003e \u003cp\u003eVisceral hypersensitivity (VH) was measured by abdominal withdrawal reflex (AWR) scores and pain threshold pressure (PTP) as described previously [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Briefly, under anesthesia with isofluorane, a flexible latex balloon with a diameter of 5 cm attached to a polyethylene tube was inserted 6 cm into the descending colon and rectum via anus and secured by taping the tube to the rat\u0026rsquo;s tail. Rats were housed in small lucite cubicles and allowed to adapt for 1 hour. Graded colorectal distention (CRD) of 20, 40, 60, and 80 mmHg was performed by rapidly inflating the balloon to constant pressure using a sphygmomanometer for 20 s followed by 2 min rest. A blinded observer observed the animal response and give a score of either 0 (no response), 1 (slight head movement followed by immobility), 2 (contraction of abdominal muscles), 3 (lifting of abdomen) or 4 (body arching and lifting of pelvic structures). Similarly, PTP was measured with CRD increased step by step at intervals of 5mmHg. The PTP was defined as the minimal pressure of the CRD when the rat showed lifting of abdomen. AWR scores and PTP were measured on the day after the completion of the 7-day WAS procedure. Each rat was tested twice and the mean AWR score and PTP from the two repeated measurements was used for each rat in the following statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence detection\u003c/h2\u003e \u003cp\u003ec-Fos immunofluorescence was determined as previously reported [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Briefly, rats were perfused with 0.01 M phosphate-buffered saline (PBS, pH 7.4) followed by 150 mL freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (4\u0026deg;C). The brains were removed immediately, post-fixed for 3 hours, and dehydrated overnight in 20% sucrose in 0.1 M phosphate buffer saline (PBS) at 4\u0026deg;C. Series of frozen coronal sections containing the CeA were cut at 30 \u0026micro;m in a cryostat and then collected into 0.01 M PBS. Endogenous active enzymes were blocked with 2% goat serum in 0.01 M PBS containing 0.5% Triton X-100 for 30 min at room temperature. The sections were incubated overnight at 4\u0026deg;C with rabbit anti-c-Fos (1:500; Abcam Trading Co. Ltd. Shanghai, China), followed by incubation with biotinylated goat anti-rabbit IgG (Hangzhou Hua\u0026rsquo;an Biotechnology Co. Ltd. Hangzhou, China) for 1 hour at room temperature. Images of the expression of c-Fos-positive neurons were obtained by a fluorescence microscope (Olympus Biological Microscope BX43, Japan). The number of c-Fos immunoreactive (IR) neurons in the CeA per unit area (0.01 mm2) was counted by using Image pro-Plus6.0 professional image analysis software (Media Cybernetics Inc, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eWestern blot was used to detect the protein expression levels of GAD65, SYN1, PSD-95 and BDNF in the CeA. Bilateral CeAs were collected according to the atlas [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and the proteins were extracted. 40\u0026micro;g of Proteins extract were separated by SDS-PAGE and then were transferred onto PVDF membranes. Subsequently, the membrane was blocked with a closure solution (5% defatted milk powder and 0.1% Tween-20 contained in 0.01 M PBS) at 4\u0026deg;C for 1 h, followed by incubation with primary antibodies: rabbit anti-Beta (1:10000; R1207-1; Huabio), rabbit anti-SYN1 (1:5000; 20258-1-AP; Proteintech), rabbit anti-PSD95(1:2000; ET1602-20; Huabio), rabbit anti-BDNF(1:10000; ET1606-42; Huabio) and mouse anti-GAD65( 1:1000;GB12562;servicebio) respectively overnight at 4℃. Then, the membranes were incubated with the secondary anti-rabbit or anti-mouse IgG antibodies for 1 hour at room temperature. Enhanced chemiluminescence staining was used and the membrane was scanned by image analysis software. Quantity One was used to measure the optical density. The amount of protein was quantified by densitometry and normalized to β-actin, an internal standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and analysed by one-way analysis of variance (ANOVA) using SPSS V.29.0 statistical software (SPSS Inc., Chicago, IL, USA). The post-hoc test of least significant difference or Tambane\u0026rsquo;s T2 test (equal variance not assumed) was used to compare mean values between groups. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEA attenuated WAS-induced VH and anxiety-like behaviors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe timeline of this part of the experiment is shown in Fig.1A. All the results shown in Fig.1 and Fig.2 are from the 4 group: sham group, WAS group, WAS+ ST25 group and WAS+ ST36 group (n=7/group). As shown in Fig.1B, the AWR scores showed no significant difference among 4 groups when the distension pressure was 20mmHg (\u003cem\u003eP\u003c/em\u003e \u0026gt;0.05) and 80mmHg (scores in all groups =4\u0026plusmn;0.00). AWR score in WAS group was significantly higher than that of sham group when the distension pressure was 40mmHg (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) and 60mmHg (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), dedicating WAS induced obvious VH. Compared with the WAS group, EA treatment at both ST25 and ST36 significantly decreased AWR score when the distension pressure was 40mmHg (\u003cem\u003eP\u003c/em\u003e=0.019 \u0026amp; \u003cem\u003eP\u003c/em\u003e=0.049 respectively). However, EA treatment did not decrease significantly AWR score when the distension pressure was 60mmHg (both \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). There was no significant difference between AWR scores in the EA+ST25 and EA+ST 36 group (all \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). Similarly, as shown in Fig.1C, PTP in WAS group was significantly Lower than that of sham group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), also dedicating WAS induced VH. Compared with the WAS group, EA treatment at both ST25 and ST36 significantly increased PTP (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). As shown in Fig.1D, Defecation frequency (DF) during the 1 hour-stress in each day in sham group was significantly lower than that of WAS, WAS+ST25 and WAS+ST36 group (all\u003cem\u003e\u0026nbsp;P\u003c/em\u003e\u0026lt;0.001), which was consistent with the fact that stress can lead to an increase in animal defecation behavior.\u0026nbsp;As shown in Fig.1E, compared with the WAS group, both EA treatments at ST25 and ST36 decreased DI significantly (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003eTo explore the effects of WAS and EA treatment in anxiety, the OFT was carried in the last day. As shown in Fig.1F-H, both central time and central entries in WAS group were significantly decreased than those in sham group (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) and defecations in WAS group were significantly increased than that in sham group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), dedicating obvious anxiety-like behaviors in WAS-induced IBS rats. Compared with the WAS group, both EA treatments at ST25 and ST36 significantly increased the central time (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), central entries (P=0.0175 \u0026amp; =0.023) and deceased defecations (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, these results indicate that WAS-induced VH, diarrhea and anxiety-Like behaviors in rats can be alleviated by EA treatment. The effects of EA at ST 25 or ST 36 have no significant difference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEA suppressed WAS-induced expression of c-Fos, GAD65, SYN1, PSD95 and BDNF in the CeA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExample images of c-Fos expression by immunofluorescence in the anterior, middle and posterior CeA of the 4 group were taken from brain sections 2.16~2.76 mm posterior to bregma (Fig.2A-B). As shown in Fig.2C, significantly more c-Fos IR neurons in the anterior, middle and posterior CeA were respectively observed in the WAS group than the sham group (all \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). EA treatment at ST25 and ST36 significantly decreased the number of Fos IR neurons in the CeA compared with the WAS group (all \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). The number of c-Fos IR neurons showed no significant difference between the EA+ST25 and EA+ST 36 group (all \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05) (Fig.2C).\u003c/p\u003e\n\u003cp\u003eThe results of western blot analysis of CeA from the 4 group are shown in Fig.2D-G. The expression of GAD65, SYN1, PSD95 and BDNF of CeA was significantly increased by WAS compared with the sham group (all \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). EA at both ST25 and ST 36 significantly inhibited GAD65 (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), SYN1 (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), PSD95 (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) and BDNF (\u003cem\u003eP\u003c/em\u003e=0.001 \u0026amp; =0.028) expression in CeA compared with the untreated WAS group. The expression of GAD65, SYN1, PSD95 and BDNF showed no significant difference between the EA+ST25 and EA+ST 36 group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWAS-induced dysfunction was alleviated by inhibiting CeA neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify the role of CeA in the development of stress-induced IBS, we used AAV9-hSyn-hM3D(Gq)-mCherry and AAV9-hSyn -hM4D(Gi)-mCherry respectively to activate and inhibit CeA neurons. The timeline of this part of the experiment is shown in Fig.3A. Immunofluorescence experiments were performed to validate the specificity of the viruses (Fig.3B). Compared with injecting AAV9-hSyn-mCherry, injecting AAV9-hSyn-hM3D(Gq)-mCherry increased c-Fos expression (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) and injecting AAV9-hSyn-hM4D(Gi)-mCherry decreased c-Fos expression (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) significantly (Fig.3C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results in Fig.3D-I were from the 4 group: AAV9-hSyn-mCherry+NS (sham) group, AAV9-hSyn-mCherry+WAS+NS (WAS) group, AAV9-hSyn-hM3D(Gq)-mCherry+WAS+CNO (WAS+CeA activation) group and AAV9-hSyn-hM4D(Gi)-mCherry +WAS+CNO (WAS+CeA inhibition) group (n=7/group). As shown in Fig.3D-I, compared with WAS group, inhibiting CeA neurons significantly decreased AWR scores (\u003cem\u003eP\u003c/em\u003e=0.032) in response to CRD of 40mmHg, increased PTP (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), decreased DI (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), increased the central time and central entries (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 \u0026amp; =0.0175) and decreased the defecations (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). And all the data indicated no significant difference between the WAS group and the WAS+CeA activation group. These results demonstrate that inhibiting CeA neurons can effectively reverse WAS-induced VH, diarrhea and anxiety-like behaviors in rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivating CeA neurons blocked the effects of EA treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis part was to investigate whether activating CeA neurons can block the effects of EA treatment. Because the effects of EA at ST 25 and ST 36 are similar in the first part of the experiment, only EA at ST 25 was observed in this part. The timeline of this part of the experiment is shown in Fig.4A. These results were from the 3 groups: AAV9-hSyn-mCherry+WAS+EA(WAS+EA) group, \u0026nbsp;AAV9-hSyn-hM3D(Gq)-mCherry+WAS+CNO（WAS+ CeA activation）group and AAV9-hSyn-hM3D(Gq)-mCherry+WAS+CNO +EA（WAS+ CeA activation +EA）group (n=7/group) .\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.4B-C, significantly more c-Fos IR neurons in the CeA were observed in the WAS+ CeA activation group and the WAS+ CeA activation+EA group respectively than WAS+EA group (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Compared with WAS+ CeA activation group, EA decreased the c-Fos expression in the CeA (\u003cem\u003eP\u003c/em\u003e=0.013). As shown in Fig.4D-I, compared with WAS+EA group, activating CeA neurons significantly increased AWR scores (\u003cem\u003eP\u003c/em\u003e=0.009) in response to CRD of 40mmHg, decreased PTP (\u003cem\u003eP\u003c/em\u003e=0.012), increased DI (\u003cem\u003eP\u003c/em\u003e=0.001), decreased the central time and central entries (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) and increased the defecations (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) in the WAS+ CeA activation+EA group. And all the data indicated no significant difference between the the WAS+ CeA activation group and the WAS+ CeA activation+EA group. These results demonstrate that activating CeA neurons can effectively block the effect of EA treatment on IBS rat.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivating or inhibiting the GABAergic neurons in the CeA respectively blocked or promoted the effects of EA treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis part was to investigate the possible role of CeA GABAergic neurons in the mediating mechanism of EA intervention. The timeline of this part of the experiment is shown in Fig.5A. Immunofluorescence experiments were performed to validate the specificity of the viruses (Fig.5B).\u003c/p\u003e\n\u003cp\u003eThe results in Fig.5C-H were from the 3 groups: AAV9-hSyn-mCherry+WAS+EA(WAS+EA) group, AAV9-GAD65-cre+AAV9-hSyn-DIO-hM3D(Gq)-mCherry+WAS+CNO+EA (WAS+GABA activation+EA) group and AAV9-GAD65-cre+AAV9-hSyn-DIO-hM4D(Gi)-mCherry+WAS+CNO+EA (WAS+GABA inhibition +EA) group (n=7/group).\u003c/p\u003e\n\u003cp\u003ecompared with WAS+EA group, activating GABAergic neurons in the CeA significantly increased AWR scores (\u003cem\u003eP\u003c/em\u003e=0.013) in response to CRD of 40mmHg, decreased PTP (\u003cem\u003eP\u003c/em\u003e=0.0072), increased DI (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), decreased the central time and central entries (both \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) and increased the defecations (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) in the WAS+GABA activation+EA group; and inhibiting GABAergic neurons in the CeA significantly decreased AWR scores (\u003cem\u003eP\u003c/em\u003e=0.041) in response to CRD of 40mmHg, increased PTP (\u003cem\u003eP\u003c/em\u003e=0.0012), decreased DI (\u003cem\u003eP\u003c/em\u003e=0.0409), increased the central time and central entries (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01=0.0371 \u0026amp; =0.0023, respectively) and decreased the defecations (\u003cem\u003eP\u003c/em\u003e=0.01) in the WAS+GABA inhibition+EA group. These findings suggest that activating or inhibiting GABAergic neurons in the CeA can impede and enhance the effects of EA treatment on IBS rats, respectively.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we observed the intervention effects of EA on stress-induced IBS rats, and explored the mediating mechanisms involving the CeA. EA suppressed the neuronal overactivity and the enhancement of neuronal plasticity induced by stress, suggesting a crucial central mechanism against stress-induced VH and anxiety. The CeA and its GABAergic neurons may serve as important target nuclei and target neurons for EA intervention. Our study, for the first time, reveals the regulatory role of the CeA in mediating the effects of acupuncture on gastrointestinal symptoms and anxiety in IBS rats.\u003c/p\u003e \u003cp\u003eIn present study, A 7-day chronic WAS induced notably VH and anxiety in rats with increased c-Fos IR neurons in CeA, consistent with previous brain imaging studies in patients with IBS[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and immunohistochemical experiment in mice[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] Our study found that EA intervention not only alleviated WAS-induced symptoms including abdominal pain, diarrhea and anxiety-like behavior but also suppressed the heightened expression of c-Fos in the CeA induced by WAS, suggesting a potential mediating role of the CeA. To confirm this point, we use chemogenetic method to induce the activation or inhibition of CeA neurons respectively. And we found that WAS-induced dysfunction was alleviated by inhibition of CeA neurons and that the effects of EA treatment was blocked by activating CeA neurons. These data suggest that EA is effective to ameliorate stress-induced VH and anxiety via inhibiting neuronal hyperactivity in the CeA.\u003c/p\u003e \u003cp\u003eNeurontrophin, especially brain derived neurotrophic factor (BDNF) expression is also regulated by stress- and activity-dependent factor and induce the change of neuronal plasticity, so it is considered a potential biomarker of many stress-related diseases including anxiety and depression [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The experiment on the effect of BDNF on synaptic morphological differentiation in hippocampus proves that in the presence of BDNF, the primary dendrites and dendritic spines in the culture medium increase significantly [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Interestingly, the trends of stress-induced change of BDNF expression in different brain area are not consistent. For example, the BDNF expression is downregulated in the hippocampus but upregulated in the amygdala in stress response [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Increased BDNF expression can enhance synaptic plasticity in the CeA, both of which contribute to stress-induced VH [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the present study, EA intervention suppressed the increasing expression of BDNF, SYN1 and PSD95 in the CeA induced by WAS. Therefore, we postulate that modulating synaptic plasticity may be a key mechanism underlying the beneficial effects of EA in ameliorating visceral pain. Notably, recent experiments have demonstrated that synaptic plasticity within the CeA is modulated by microglia after exposure to chronic stress [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, in further research, exploring whether microglia within the CeA could be considered as potential targets for acupuncture to intervene in IBS deserves attention.\u003c/p\u003e \u003cp\u003eIn the current study, we observed that WAS induced significant higher expression of GAD 65 in CeA, indicating increased GABA synthesis and activated GABAergic neurons. EA decreased the level of GAD65 expression in CeA, suggesting the possible mediating role of GABAergic neurons in the CeA. To confirm this point, we use chemogenetic method again to induce the activation and inhibition of GABAergic neurons in the CeA respectively. And we found activating and inhibiting GABAergic neurons in the CeA can, respectively, impede and enhance the effects of EA treatment on IBS rats. These data suggest that EA is effective to ameliorate stress-induced VH and anxiety via inhibiting the hyperactivity of GABAergic neurons in the CeA. However, GABAergic neurons in CeA are involved in several different inhibitory circuits or pathways inside or outside amygdala [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and can act on the GABAA, GABAB and GABAC receptors [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Further investigation is needed to determine which specific inhibitory pathways are affected by EA and the mechanisms through which EA exerts its effects. Additionally, it is crucial to explore whether a specific GABA receptor is involved in mediating the regulatory effects of EA.\u003c/p\u003e \u003cp\u003eIn summary, our results demonstrated that inhibiting the excessive activity of neurons in the CeA, especially the excessive activity of GABAergic neurons, and suppressing enhanced synaptic plasticity in the CeA are potential mechanisms for the positive effects of EA in the treatment of IBS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Shandong Province (Grant numbers:\u0026nbsp;ZR2021MH199, ZR2020MC075,\u0026nbsp;ZR2021MC142\u003cstrong\u003e)\u0026nbsp;\u003c/strong\u003eand the National Natural Science Foundation of China (32170496)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRongxin Weng, Qinghui Wei, Zepeng Wang and Xinyu Li performed the experiments, analyzed the data, and contributed to study design and manuscript writing. Yuanyuan Li, Xuehan Sun, Yuxue Wang and Xiusong Wang performed the behavioral studies and contributed to data analyzing. Feng He and Haiji Sun supervised the study, analyzed data and wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the results of this research can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Animal Experimental Ethics Committee of Shandong Normal University (reference no. AEECSDNU2020003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLovell RM, Ford AC (2012) Global prevalence of and risk factors for irritable bowel syndrome: a meta-analysis. Clin Gastroenterol Hepatol 10(7):712-721. https://doi.org/10.1016/j.cgh.2012.02.029\u003c/li\u003e\n\u003cli\u003eDrossman DA (2016) Functional gastrointestinal disorders: history, pathophysiology, clinical features, and Rome IV. Gastroenterology 150(6): 1262-1279.https://doi.org/10.1053/j.gastro.2016.02.032\u003c/li\u003e\n\u003cli\u003eBai Y, Chen YB, Qiu XT, Chen YB, Ma LT, Li YQ et al (2019) Nucleus tractus solitarius mediates hyperalgesia induced by chronic pancreatitis in rats. 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J Cancer Prev 28(3):93-105.https://doi.org/10.15430/JCP.2023.28.3.93\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Irritable bowel syndrome (IBS), Electroacupuncture (EA), Central amygdala (CeA), Visceral hypersensitivity (VH), Anxiety","lastPublishedDoi":"10.21203/rs.3.rs-3876166/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3876166/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVisceral hypersensitivity (VH) and anxiety are the main characteristics of irritable bowel syndrome (IBS) patients. Acupuncture has been demonstrated to be effective as a non-pharmacological approach for treating IBS. The central amygdala (CeA) is a key brain nucleus closely related to mediate integration of abdominal pain, gastrointestinal motility and anxiety. In the present study, we investigated whether and how the CeA mediate the protective effects of electroacupuncture (EA) against VH and anxiety in water avoidance stress (WAS)-induced IBS rats. Visceral sensitivity was assessed via abdominal withdrawal reflex and diarrhea index. Anxiety-related behavior was measured via open field test. The expression of c-Fos, GAD65, SYN1, PSD95 and BDNF in CeA were measured by using immunofluorescence or western blot. The chemogenetic method was used to activate or inhibit the selected target neurons. We found that EA attenuated WAS-induced gastrointestinal symptoms and anxiety-like behaviors with reduced expression of c-Fos, GAD65, SYN1, PSD95 and BDNF in the CeA. WAS-induced dysfunction was alleviated by inhibiting CeA neurons and the effects of EA treatment were counteracted by activating CeA neurons. Moreover, activating and inhibiting gamma-aminobutyric acidergic (GABAergic) neurons in the CeA can impede and enhance the effects of EA treatment, respectively. These data suggest that EA is effective to ameliorate stress-induced VH and anxiety via alleviating neuronal hyperactivity in the CeA, particularly targeting the hyperactivity of GABAergic neurons in the CeA, and suppressing enhanced synaptic plasticity in this region.\u003c/p\u003e","manuscriptTitle":"Mechanisms of Electroacupuncture in Alleviating Stress-induced Irritable Bowel Syndrome in Rats: The Role of the Central Amygdala","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-30 18:07:58","doi":"10.21203/rs.3.rs-3876166/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4af1708b-0d15-4c3d-8f7d-45a832668aeb","owner":[],"postedDate":"January 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-27T05:39:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-30 18:07:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3876166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3876166","identity":"rs-3876166","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}