CHRNA3⁺ Nociceptors Prime the Cutaneous Sensory Interface to Enhance Electroacupuncture Analgesia

CHRNA3⁺ Nociceptors Prime the Cutaneous Sensory Interface to Enhance Electroacupuncture Analgesia | 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 CHRNA3⁺ Nociceptors Prime the Cutaneous Sensory Interface to Enhance Electroacupuncture Analgesia Wenjie Xu, Dingdan Zhang, Yuanwei Tang, Ying Wang, Zijie Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6684432/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Recent evidence determined that acupoints frequently overlap with regions of referred somatic hypersensitivity induced by visceral disease, a phenomenon known as acupoint sensitization. This state is typically characterized by sensory hypersensitivity and functional enhancement, often accompanied by superior therapeutic outcomes following acupuncture. However, the neurobiological mechanisms that prime acupoints for enhanced responsiveness remain poorly understood. Methods Using a rat model of TNBS intracolonic injection-induced colitis and relative acupoint sensitization, we investigated the role of CHRNA3⁺ mechanoinsensitive nociceptors (MINs), a subclass of silent C-fiber neurons, in modulating the acupoint sensory interface and electroacupuncture (EA) responsiveness. Behavior tests, neuroanatomical tracing, in situ hybridization, pharmacological blockade, and chemogenetic silencing were employed to assess the involvement of CHRNA3⁺ MINs in the onset of acupoint sensitization. Colonic distension-evoked visceral motor reflex and spinal local field potential recording were utilized to evaluate the contribution of CHRNA3⁺ MINs in the acupuncture-induced analgesic effect. Results We found that CHRNA3⁺ MINs are primarily C nociceptors co-expressing TrkA, pERK, and PIEZO2, and that they innervate both the colon and lumbar skin (BL25 acupoint region) via axonal bifurcation. Colitis significantly activated CHRNA3⁺ nociceptors via the NGF–TrkA/pERK/PIEZO2 pathway, converting them from mechanoinsensitive to mechanosensitive. This activation correlated with increased plasma extravasation and mechanical allodynia at BL25. Pharmacological inhibition of CHRNA3⁺ MINs via pERK blockade or chemogenetic silencing reduced mechanical hypersensitivity of BL25 and attenuated the analgesic effects of EA on both visceral pain and spinal sensitization. Conclusion Our findings reveal that CHRNA3⁺ silent nociceptors dynamically prime and reshape the sensory interface of the colitis-induced sensitized acupoint BL25, facilitating both pathological hypersensitivity and therapeutic responsiveness. This study establishes a mechanistic link between visceral dysfunction, acupoint functional plasticity, and EA-induced therapeutic neuromodulation. Acupoint sensitization Mechanoinsensitive nociceptors Silent nociceptors Electroacupuncture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Acupuncture has been practiced for millennia, yet the anatomical basis of acupoints remains elusive. Numerous studies have compared acupoints to adjacent non-acupoint regions, reporting subtle differences in sensory nerves [ 1 ] , blood vessels, lymphatic vessels [ 2 ] , connective tissues [ 3 ] , tissue space areas [ 4 ] , and mast cells [ 5 ] . The absence of a strikingly anatomical signature raises a critical question: are acupoints fixed landmarks or dynamic, state-dependent sensory interfaces? Two longstanding challenges in acupuncture research compound this question. First, sham or “non-acupoint” controls often elicit effects comparable to true acupoints, undermining their validity. Second, the “wide-pan-acupoint” phenomenon, therapeutic benefit from needling outside traditional points, suggests that acupoint efficacy depends more on the local sensory state than on precise location. Together, these observations argue for reframing acupoints as plastic sensory interfaces, reshaped by disease, particularly visceral diseases, rather than immutable anatomical sites. Clinically, needling hypersensitive regions, such as Ashi and trigger points, consistently performs promising therapeutic outcomes, highlighting the potential role of sensory plasticity in acupoint function. Acupoint sensitization is a recently characterized functional state in which somatic regions corresponding to acupoints exhibit sensory abnormalities under pathological conditions such as visceral disease. This state is defined by two key features: sensory hypersensitivity (e.g., mechanical allodynia) and heightened responsiveness to therapeutic stimuli like acupuncture [ 6 – 13 ] . Although these sensitized areas frequently overlap with regions of visceral referred pain [ 6 – 13 ] , the core concept of acupoint sensitization emphasizes the resulting sensory abnormality and functional plasticity, which optimizes these sites for receiving and transducing therapeutic stimuli. Neurogenic inflammation is a key pathological driver of this sensitized state. It is initiated when activated sensory terminals release neuropeptides, stimulating local non-neuronal cells like mast cells and macrophages. This interaction establishes a self-amplifying feedback loop that primes the acupoint, enhancing its responsiveness to acupuncture. However, the precise mechanism through which visceral disease engages this neuroinflammatory cascade to modulate the acupoint's sensory interface remains undetermined.Peripheral sensory neurons, commonly referred to as nociceptors, emerging as critical players for acupuncture manipulation. Typically, C-nociceptors, comprised of peptidergic and non-peptidergic subtypes, have testified the essential role in the onset of acupoint sensitization [ 14 – 18 ] . Among these, silent nociceptors, also referred to as mechanoinsensitive nociceptors (MINs), have been implicated in the initiation of pain onset and exacerbation [ 19 – 22 ] , and itch [ 23 ] . These neurons, innervate visceral organs and somatic tissues [ 19 , 21 , 24 ] , are typically unresponsive to mechanical stimuli under normal conditions but become sensitized and mechanosensitive in the presence of inflammatory mediators, such as nerve growth factor (NGF). Recent study has identified the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3), a subunit of the nicotinic acetylcholine receptor, as a specific molecular marker of MINs in mice [ 20 , 21 ] . CHRNA3 neuron densely innervates somatic tissues and viscera and accounts for 40% of the peptidergic C-nociceptors, contributes to secondary mechanical hyperalgesia development [ 20 , 21 ] . Electrophysiological evidence reveals the “awaken” features of CHRNA3 + neurons, which convert from mechano-insensitive to mechano-responsive following NFG through TrkA/pERK/PIEZO2 signaling pathway. Given this mechano-responsiveness similarity, we propose that CHRNA3 + MIN mediated both the mechanical allodynia of sensitized acupoints and the amplification of sensory input during acupuncture. In this study, we identify CHRNA3⁺ neurons as a distinct subset of C-fiber nociceptors in rats, co-expressing with both peptidergic and non-peptidergic nociceptors. These neurons innervate visceral organs and homo spinal segmental somatic tissues, including the colon and the BL25 acupoint region, via axonal bifurcation, providing a structural substrate for viscerosomatic integration. Using a colitis-induced acupoint sensitization model, we demonstrate that CHRNA3⁺ MINs are activated by colonic inflammation and contribute to colitis-induced referred mechanical hypersensitivity at the BL25 acupoint, a classical site used in acupuncture for gut-related dysfunctions. Chemogenetic inhibition of CHRNA3⁺ MINs alleviated acupoint hyperalgesia and significantly reduced the efficacy of electroacupuncture (EA) in both behavioral and spinal neuronal readouts. These findings suggest that CHRNA3⁺ MINs not only mediate pathological sensitization but also act to prime and reshape the sensory interface of the acupoint, rendering it more responsive to therapeutic stimulation. This study presents a novel neurobiological mechanism through which silent nociceptors contribute to both the sensitization and functional plasticity of acupoints. Our findings bridge somatic sensory interface reshaping caused by visceral disease with acupoint and acupuncture effect, offering a mechanistic basis for understanding how nociceptor-mediated sensory interface prime acupoint function enhancing its modulatory effect. Materials and Methods Animals Male Sprague-Dawley rats (220 ± 10 g) were purchased from Charles River (Beijing; license: SCXK-[Beijing]-2021-0011) and housed at the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (CACMS). Rats were maintained under standard laboratory conditions (12 h light/dark cycle, 24 ± 0.5°C, 60%–70% humidity) and allowed to acclimate for one week before experimentation. All procedures were approved by the Institutional Animal Care and Use Committee of CACMS (Ethics No. Y2022-03-10-06). TNBS-Induced Colitis Model Colitis was induced as previously described [ 25 ] . After a 24-hour fast, rats were anesthetized with 2% isoflurane, and TNBS (100 mg/kg in 50% ethanol, 2:1 ratio, Sigma-Aldrich) was administered intracolonically via a 30-gauge catheter inserted 6–7 cm from the anus. Rats were held vertically for 5 min post-injection to ensure retention. Control rats received equal volumes of saline or 50% ethanol. Animals were monitored daily for body weight, general condition, and signs of colitis. Disease Activity Index (DAI) and Tissue Damage Index (TDI) Colitis severity was assessed using the DAI and TDI scoring systems [ 26 ] . DAI was calculated as the average of three parameters: weight loss (0–4), stool consistency (0 = normal, 2 = loose, 4 = diarrhea), and rectal bleeding (0 = none, 2 = occult, 4 = gross). TDI was assessed on H&E-stained colon sections by two blinded pathologists, evaluating inflammatory infiltration, crypt loss, ulceration, and mucosal damage. Images were captured using a microscope (BX53, Olympus, Tokyo, Japan) and evaluated with Image J software (US National Institutes of Health, Bethesda, MD). Evans Blue Plasma Extravasation Skin plasma protein extravasation following Evans Blue (EB) administration in colitis rats was conducted as previously described [ 27 ] . Briefly, rats were anesthetized with 2% isoflurane at 7 days after colitis operation, and Evans Blue (50 mg/kg in saline, Sigma) was injected into the lateral tail vein. After 30–60 min, PE points were mapped and counted. Hair was removed from the dorsal and hindlimb regions 24 h prior using depilatory cream (CP-8000, Codos, China). Mechanical Sensitivity Testing Mechanical thresholds at BL25 were measured using an electronic von Frey apparatus (ALMEMO 2450, AHLBORN) as previously described [ 27 ] . Rats were acclimated to a restraining pocket for 2 days and habituated for 30 min before each test. Measurements were taken on days 1, 3, and 7, and the mean of three trials was used. AAV virus injection To investigate whether dorsal root ganglion (DRG) neurons innervate both the colon and the BL25 acupoint region via axonal bifurcation, scAAV2/9-hSyn-EGFP (2.0 × 10¹² VG/mL, PT-2315, Shumi) and scAAV2/9-hSyn-mCherry (2.0 × 10¹² VG/mL, PT-3975, Shumi) were separately injected into the colonic wall and BL25 region. For intracolonic injections, a 1 cm midline abdominal incision was made at the rat under isoflurane anesthesia, and 10 µL of EGFP virus was injected between the muscularis externa and serosa at 5–6 evenly spaced sites (~ 2.5 cm total length) using a microsyringe under microscopic guidance. This multi-site, low-volume-per-site strategy was designed to maximize coverage while minimizing local tissue stress. For intracutaneous injections at BL25, a total of 10 µL of mCherry virus was delivered across 5–8 evenly distributed points in the bilateral BL25 region. Ten days after injection, L6–S1 DRGs were harvested, sectioned, and processed for colocalization analysis. To selectively inhibit colonic CHRNA3⁺ neurons, rAAV-Chrna3-ZsGreen (2.6 × 10¹² VG/mL) and rAAV-Chrna3-hM4D(Gi)-ZsGreen (Braincase) were injected into the colonic wall of the rat. A 1 cm incision was made under isoflurane anesthesia, and 10 µL virus was injected between the muscularis externa and serosa across 5–6 sites (~ 2.5 cm span). Colitis modeling was conducted 21 days after AAV injection to allow for sufficient transgene expression. Behavioral and electrophysiological experiments were conducted 7 days after colitis modeling (28 days after virus injection). The timeline was consistent for all chemogenetic experiments. Immunofluorescence and In Situ Hybridization Rats were perfused with PBS followed by 4% paraformaldehyde. DRG, skin, colon, and spinal tissues were harvested, cryoprotected in 30% sucrose, embedded in OCT, and cryosectioned at 20 µm. Sections were blocked with 10% serum and incubated overnight at 4°C with primary antibodies, followed by fluorescent secondary antibodies and DAPI. Confocal imaging was performed using an FV1000 microscope (Olympus, Tokyo, Japan). The following primary antibodies were used: CHRNA3, (Invitrogen, PA5-77501, 1:200), PIEZO2 (Novus, NBP2-58161, 1:50), TrkA (R&D, AF1056, 1:200), TrkA (NOVUS, AF1494, 1:200), p-ERK (Santa Cruz, sc-7383, 1:200), NeuN (Sigma RNAscope Multiplex Fluorescent Reagent Kit V2 (Cat. No. 323110) was used to perform in situ hybridization with chrna3 (527051-C1, ACD) and PIEZO2 (549741-C3, ACD) probes according to the manufacturer’s protocol. Western blot assay Western blot assay Seven days after colitis modeling, the bilateral lumbar L6-S1 DRG was collected and homogenized in ice-cold RIPA lysis buffer (89900, Thermo Scientific) with Halt Protease Phosphatase Inhibitor (5872s, CST, 1:500). Protein concentrations were measured with a Pierce BCA Protein Assay Kit (23225, Thermo Scientific). Equal amounts of the proteins sample were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes. Proteins samples were then incubated with antibodies for rabbit anti-ERK1/2 (A4782, Abclonal, 1:2000), rabbit anti-pERK1/2 (AP0472, Abclonal, 1:1000), rabbit anti-NGF (Abcam, Abclonal, ab52918, 1:2000), and β-actin (XAb-A, X-blot) overnight at 4℃, followed by incubation with the relative secondary antibodies. The interested bands were visualized using an ECL kit (170–5060, BIO-RAD) and imaged with iBright1500 (Invitrogen). The β-actin served as the loading control and intensity of the selected bands was analyzed using Image J. U0126 Treatment U0126 (5 mg/kg/day, i.p.) was administered to rat for 7 days after colitis induction. Stock solution (50 mg/mL in DMSO) was diluted to 5 mg/mL in 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. Electroacupuncture (EA) Rats were placed in a prone position on a heating pad and anesthetized with inhalation of 2% isoflurane. The bilateral BL25 acupoints are located at the depression lateral to the lower border of the spinous process of the fourth lumbar vertebra. The needles (0.25×25 mm, Suzhou Medical Appliance Factory, Suzhou, China) were inserted into the bilateral BL25 point at a depth of ~ 3 mm. EA were delivered using a stimulator (STG4000, Warner Instruments, U.S.A) with the following parameters [ 26 ] : 2 Hz frequency, 1 mA intensity, for a duration of 10 min. Visceromotor Reflex (VMR) Recording Visceromotor response (VMR) was recorded as an indicator of Visceral hyperalgesia (VH) by colorectal distension (CRD) which was aroused by using a custom-made balloon [ 26 ] . After mice were anesthetized with 2% isoflurane, a lubricated polyethylene balloon (1cm diameter) was inserted transanally into the colorectum approximately 0.5-1 cm from the anal verge. The external portion was secured to the tail with tape. Colorectal distension was produced by rapidly inflating the balloon to a constant pressure of 60 mmHg. The pressure was maintained for 20 seconds and allowed 5 min for recovery between each inflation. EMG spikes were recorded by the PowerLab Data Recording & Analysis System (ADIstruments Pty Ltd). Spinal LFP Recording The experimental setup for in vivo spinal LFP recording was similar to our previous studies.5–7 Briefly, the rats were anesthetized with urethane (1.2 g/kg, i.p.). The lumbar spinal cord was exposed in anesthetized rats and the dura mater was partially removed at the recording segments (L6-S1). The paralyn-coated tungsten microelectrode (3 mΩ, Frederick Haer Company, Brunswick, ME, USA) was inserted into the superficial dorsal horn (200–500 µm below the surface) at L6 spinal segment. The left sciatic nerve was exposed and placed on the hook electrode to deliver the test stimulus (5 mA, 0.2 msec, 1 test/1 min), evoking the spinal LFP. A real-time, computer-based data acquisition and processing system (CED Spike 2, Cambridge, UK) was used to collect analog data. Raw data was collected at a sampling rate of 1000 Hz. The data stream was amplified and then filtered (0.1–100 Hz, model DAM80; World Precision Instruments, Sarasota, FL, USA), and artifacts of stimulation were removed online by a notch filter (IIR filters of the CED 1401 data-acquisition system). LFP was examined before EA (baseline, 10 min) and at 0–30 min after EA application. Based on the conduction velocity, LFPs that correspond to activated A- and C-fibers can be distinguished. In comparison to the large A-LFP, the C-LFP exhibits a longer latency (90–130 msec) and smaller amplitude. Offline modulus measurements of Spike2 software were used to analyze the area under curves (AUC) of C-LFP. A comparison was made between pre- and post-EA conditions within and between the groups after normalizing LFP to pre-EA baseline values in each animal. Statistical Analysis Sample sizes (n = 6–8 per group) were determined based on established standards in the field for similar models and endpoints, which consistently yield large effect sizes. This approach aligns with the ethical goal of minimizing animal use while ensuring robust detection of biological effects. The GraphPad Prism (version 8.0, GraphPad Software, San Diego, CA, USA) software was used for statistical analysis. The normality of data was checked using the Shapiro‒Wilk test. Data are presented as mean ± SEM. A paired or independent t-test was used to analyze data within two groups. One-way or two-way ANOVA with post hoc Bonferroni test was utilized for group comparison. Significance was set at P < 0.05. Results CHRNA3⁺ nociceptors innervate peripheral tissues and prime acupoints for colitis-induced sensitization Concerning previous studies of CHRNA3 neurons in pain conducted in mice, we herein examined the innervation patterns of CHRNA3 + mechanoinsensitive nociceptors (MINs) in rats for the first time. Using immunofluorescence, we observed that CHRNA3⁺ MINs innervate the lumbar skin, colon, L6 dorsal root ganglion (DRG), and lamina I of the L6–S1 spinal dorsal horn (Fig. 1 A), indicating their widespread peripheral and central connectivity. To determine the nociceptor subtype identity of CHRNA3⁺ neurons, we performed co-staining with markers for A-fiber neurons (NF200, TrkB), C-fiber markers (Peripherin), and subtype indicators for peptidergic (CGRP⁺) and non-peptidergic (IB4⁺) populations. CHRNA3⁺ neurons showed minimal overlap with A-fiber markers (NF200: 7.94%; TrkB: 8.37%) but significant co-expression with C-fiber markers (Peripherin: 50.29%), IB4 (72.69%), and CGRP (44.55%) (Fig. 1 B–C). These data confirm that CHRNA3 is predominantly expressed in C-fiber nociceptors, convering both peptidergic and non-peptidergic subtypes. CHRNA3⁺ MINs Mediate Colitis-Induced Acupoint Sensitization To assess the functional involvement of CHRNA3⁺ MINs in acupoint sensitization, we employed a TNBS-induced colitis model known to produce referred somatic hypersensitivity [ 9 , 28 ] . Evans blue (EB) dye was injected intravenously to visualize sites of neurogenic inflammation, marked by plasma extravasation (PE) points [ 29 , 30 ] . Colitis significantly increased the number of PE points in the abdominal and lumbar regions compared to saline controls (Fig. 2 A–C), and these overlapped with classical acupoints such as BL25 (Fig. 2 C-b), consistent with previous study about the sensitized acupoints under colitis. Inhibition of CHRNA3⁺ MINs with U0126, a pERK inhibitor, significantly reduced the number of PE points. Given that mechanical hypersensitivity is a hallmark of sensitized acupoints, colitis-induced sensitized acupoint BL25 was selected and mechanical thresholds was assesedusing electronic von Frey testing. Colitis significantly reduced mechanical thresholds at BL25, indicating mechanical allodynia, which was ameliorated by U0126 treatment (Fig. 2 E–F). These findings suggest that activation of CHRNA3⁺ MINs is necessary for the onset of acupoint hypersensitivity and mechanical sensitization during colitis. To investigate whether axonal bifurcation of CHRNA3⁺ neurons mediate the interaction between the colon and the BL25 acupoint region, scAAV2/9-hSyn-EGFP and scAAV2/9-hSyn-mCherry were injected separately into the colonic wall and the BL25 region, respectively. Co-staining with CHRNA3 and NeuN revealed that approximately 8.55% of total NeuN⁺ DRG neurons were triple-labeled with GFP, mCherry, and CHRNA3 (Fig. 2 D–E). These findings indicate that a subset of CHRNA3⁺ neurons simultaneously innervate both the colon and the skin, providing a neuroanatomical basis for viscerosomatic crosstalk. Additionally, we examined whether CHRNA3⁺ MINs contribute to tissue injury during colitis. TNBS significantly increased the colonic tissue damage index (TDI) and disease activity index (DAI) (Supplementary Fig. 1). Interestingly, U0126 reduced TDI scores but not DAI, implying that CHRNA3⁺ MINs may selectively modulate local tissue injury responses without altering overall disease burden. CHRNA3⁺ MIN Activation Is Driven by TrkA/pERK/PIEZO2 Signaling Previous work in mice identified TrkA/pERK/PIEZO2 signaling as a key pathway underlying silent nociceptor activation [ 21 ] . Using in situ hybridization and immunofluorescence in rat L6 DRG, we confirmed that 47.33% of CHRNA3⁺ neurons co-expressed both TrkA and PIEZO2, while 25.03% of TrkA⁺ and 45.67% of PIEZO2⁺ neurons were triple-labeled (Fig. 3 B–C; Supplementary Figure S2). We next investigated whether colitis activates CHRNA3⁺ MINs through this pathway. Immunostaining showed a marked increase in pERK⁺ CHRNA3⁺ neurons in the DRG of colitis rats compared to saline controls (Fig. 3 D–E). U0126 treatment significantly reduced this activation. Similarly, pERK co-localization with TrkA⁺ and PIEZO2⁺ neurons increased after colitis and was suppressed by U0126 (Fig. 4 ). To determine whether NGF is a potential upstream driver [ 21 ] , we assessed NGF expression in the colon, DRG, and BL25 skin. NGF was significantly upregulated in colon and DRG, as well as an elevation trend in skin following colitis (Supplementary Fig. 2), supporting a role for NGF–TrkA–pERK–PIEZO2 signaling in CHRNA3⁺ MIN activation. These data strongly support a mechanism in which colitis activates CHRNA3⁺ MINs via NGF-dependent signaling, promoting their conversion from a mechanoinsensitive to a mechanoresponsive state—thus priming the acupoint for heightened sensory input. Chemogenetic Inhibition of CHRNA3⁺ MINs Alleviates Acupoint Hyperalgesia To directly assess the contribution of CHRNA3⁺ MINs to mechanical hypersensitivity, we constructed an AAV vector expressing inhibitory DREADD (hM4Di) under the Chrna3 promoter (AAV-Chrna3-hM4Di-ZsGreen) and confirmed that ~ 91.67% of CHRNA3⁺ neurons expressed the reporter three weeks post-intracolonic injection (Fig. 5 A–C). CNO administration in AAV-hM4Di-transduced rats significantly increased mechanical thresholds at BL25 compared to pre-CNO and control virus (AAV-ZsGreen) animals, indicating attenuation of colitis-induced mechanical hypersensitivity (Fig. 5 D–E). These results functionally confirm that CHRNA3⁺ MIN plays a role in impacted acupoint microenviroment, which then primed the interface and sustained the sensitized acupoint state for pathological mechanical responses. CHRNA3⁺ MINs Facilitate Electroacupuncture-Induced Analgesia To determine whether CHRNA3⁺ MINs enhance acupuncture signaling, we assessed the analgesic effects of electroacupuncture (EA) at BL25 on colorectal distension (CRD)-evoked visceral motor reflexes (VMRs) [ 26 ] . EA significantly reduced the EMG area under the curve (AUC) during CRD in colitis rats, while CNO-induced silencing of CHRNA3⁺ neurons attenuated this inhibitory effect (Fig. 6 A–C), suggesting that CHRNA3⁺ MINs facilitate EA-induced analgesia. To evaluate central effects, we recorded spinal cord C-local field potentials (C-LFPs) in response to sciatic nerve stimulation. Colitis elevated C-LFP amplitudes compared to Sham, reflecting spinal sensitization [ 26 ] . EA at BL25 suppressed C-LFPs at 5, 10, and 15 min, but this suppression was abolished by CHRNA3⁺ neuron silencing (Fig. 7 F–G). Together, these data determined that CHNRA3 + MIN is involved in acupuncture’s signals transduction, suggesting CHRNA3⁺ MINs not only primes peripheral acupoints but also enables EA-driven inhibition of spinal nociceptive transmission. Discussion Increasing evidence suggests that acupoints are associated with disease-induced referred somatic pain, a phenomenon known as acupoint sensitization [ 31 – 33 ] . However, the neural basis of acupoint sensitization, and how it mediates and transmits acupuncture signals, remains poorly understood. In this study, we identified CHRNA3, a known marker of mechanoinsensitive nociceptors (MINs) in mice, as being predominantly expressed in C-fiber nociceptors in rats, with widespread innervation of peripheral tissues. We further demonstrated that activation of CHRNA3⁺ MINs contributes to colitis-induced acupoint sensitization and mechanical hypersensitivity at the BL25 acupoint via the TrkA/pERK/PIEZO2 signaling pathway. Notably, these neurons appear to prime the peripheral sensory field, enhancing the responsiveness of acupoints to both nociceptive and therapeutic stimuli, such as EA. At the same time, colitis-driven CHRNA3⁺ MIN activation reshapes the acupoint sensory interface, converting it into a hyperexcitable state that facilitates signal amplification. Accordingly, chemogenetic inhibition of CHRNA3⁺ MINs not only alleviated mechanical hyperalgesia at sensitized BL25 but also attenuated the analgesic effects of EA on colitis-related visceral hypersensitivity. These findings highlight a dual role for CHRNA3⁺ MINs in priming and reshaping acupoint function, thereby modulating both pathological sensitization and acupuncture-induced analgesia (Fig. 8 ). Nociceptors Mediate Acupoint Sensitization The concept of acupoint sensitization has gained attention in recent decades, supported by clinical and preclinical observations that many acupoints overlap with regions of referred somatic hypersensitivity associated with visceral disease or tissue injury. Sensory hypersensitivity and functional enhancement are hallmark features of this phenomenon. Notably, mechanical allodynia is considered the primary manifestation of acupoint sensitization [ 6 – 9 ] , aligning with the classical concept of "selecting the painful point as the acupuncture point" described in the Jing Jin chapter of the Miraculous Pivot . In this study, we confirmed that regions of plasma extravasation overlapped with colitis-related acupoints, supporting the pathological relevance of referred hypersensitivity [ 9 , 34 ] . Meanwhile, although both the colon and the BL25 acupoint are innervated by the same spinal segments (L6–S1), we herein demonstrate that axonal bifurcation may serve as a potential neuroanatomical basis for colitis-induced acupoint sensitization. While multiple studies have explored anatomical substrates contributing to acupoint function, including sensory nerves [ 1 ] , blood vessels, lymphatic vessels [ 2 ] , connective tissues [ 3 ] , tissue space areas [ 4 ] , and mast cells [ 5 ] , no single structure clearly distinguishes acupoints from adjacent non-acupoint regions. This has led to a shift in understanding acupoints not as fixed structures, but as primed sensory interfaces at the somatic area, dynamically modulated by pathophysiological states and capable of integrating acupuncture inputs [ 2 , 35 ] . Acupoints contain a complex neuroimmune microenvironment involving nociceptors, immune cells, blood vessels, and autonomic terminals. Among these, nociceptors are thought to serve as primary transducers of acupuncture signals, and increasing evidence supports their essential role in acupoint sensitization and EA-induced neuromodulation [ 14 – 16 ] . In a rat model of knee osteoarthritis, Zhang et al. found that sensitized ST35-associated DRG neurons displayed enhanced excitability and Ih-current density in C-fibers, but not Aδ-fibers [ 17 ] . Other studies have shown that acupoint sensitization is associated with C-fiber-mediated neurogenic inflammation, evidenced by elevated CGRP and SP levels in the skin [ 29 , 30 , 36 ] . Our findings extend this by demonstrating that CHRNA3⁺ MINs, despite only partial overlap with CGRP (∼40%), play a critical role in initiating acupoint sensitization. Chemogenetic inhibition of CHRNA3⁺ neurons reduced colitis-induced plasma extravasation and mechanical allodynia, supporting their functional involvement. Recent studies reveal the immune cells and even the immune process were modulated by the nociceptor-driven neuropeptide such as CGRP and SP, which produces dual role in different pathological contexts [ 37 ] . Whether and how CHRNA3 + MINalso engage with immune cells to shape acupoint sensory interface remains an open question for future studies. Silent Nociceptor Activation Primes the Acupoint Sensory Interface C-fiber nociceptors comprise a diverse population with distinct molecular and functional properties. Single-cell RNA sequencing has revealed at least 18 transcriptomic clusters [ 38 ] . These neurons can be broadly categorized into peptidergic (e.g., CGRP⁺, SP⁺) and non-peptidergic (e.g., IB4⁺) subtypes. CHRNA3 is predominantly co-expressed with Peripherin and colocalizes with both peptidergic and non-peptidergic C-nociceptors (Fig. 1 B–C). Functionally, C-nociceptors are further distinguished by their responsiveness to thermal, chemical, or mechanical stimuli, which may shift under pathological conditions [ 39 – 42 ] . Silent nociceptors represent a unique subtype that are typically unresponsive to mechanical stimuli under normal conditions but acquire mechanosensitivity following inflammation or injury via the TrkA/pERK/PIEZO2 axis [ 21 ] . This "awakening" process is well documented across species: approximately 15–20% of cutaneous C-fibers in humans are silent nociceptors, with even higher proportions (30–90%) reported in visceral and articular tissues in rodents [ 19 , 21 , 43 – 45 ] . In a mouse model of colitis, the proportion of mechanosensitive C-fibers rose from 34% to 53%, while silent nociceptors decreased from 27% to 13%, implying their conversion to an active state. Recent work also highlights the role of MINs in secondary mechanical hypersensitivity in joint inflammation [ 20 ] . The Lechner group identified CHRNA3 as a reliable molecular marker for MINs in mice and showed that their activation is driven by NGF through the TrkA/pERK/PIEZO2 signaling cascade [ 21 ] . Subsequent studies of Lechner group identified TMEM100 as a key downstream molecular switch regulating silent nociceptor activation and mediate secondary hyperalgesia [ 20 ] . Based on these insights, we propose that CHRNA3⁺ MINs play a dual role in both the development of mechanical allodynia at sensitized acupoints and the amplification of acupuncture signals. Consistent with this model, our data confirmed co-expression of CHRNA3 with TrkA, pERK, and PIEZO2 in rat DRG neurons and demonstrated that CHRNA3⁺ MINs are activated by colitis. Despite observing only a trend toward increased NGF in the skin in this study, NGF levels in colon and DRG were significantly elevated under colitis condition. This suggests spatial heterogeneity in NGF regulation across sensory neurons, the primary site of pathology, and their referred peripheral terminals. Plus, we also observed that participation of NGF-TrkA/pERK/PIEZO2 signaling in acupoint sensitization, suggested by the increased co-expression portion of TrkA and PEIZO2 with pERK under TNBS and reversed upon U0126 administration. Chemogenetic silencing of these neurons abolished their mechanical conversion and reduced both acupoint sensitization and mechanical hypersensitivity, supporting their crucial role in priming the sensory interface. However, because we did not assess CHRNA3 expression in autonomic ganglia, including the pelvic ganglion, or in the nodose ganglion (which is critical for interoceptive signaling) during our chemogenetic viral experiments, the potential contributions of CHRNA3⁺ neurons in these ganglia remain unclear and warrant further investigation. CHRNA3⁺ MINs Amplify Acupuncture Signaling Functionally, CHRNA3⁺ MIN activation appears to enhance the analgesic effects of acupuncture. Silencing CHRNA3⁺ neurons attenuated EA-induced inhibition of visceral pain responses and spinal neuronal hyperactivation. Previous studies have shown that C-nociceptors are essential for acupuncture effects: for example, TRPV1 knockout mice exhibit impaired EA-induced analgesia [ 15 ] ,, and Prokr2 sensory neurons mediate EA-driven anti-inflammatory responses [ 46 ] . However, most prior work has focused on how somatic sensory input generates downstream effects. In contrast, our study reveals a novel mechanism whereby CHRNA3⁺ MINs prime the sensory interface, rendering acupoints more responsive to mechanical and EA stimulation. Increasing evidence demonstrated the superior therapeutic effect when targeting sensitized acupoints, such as the management of chronic musculoskeletal and non-musculoskeletal pain [ 10 – 12 ] , bronchial asthma [ 47 ] , and allergic rhinitis [ 48 ] , but the mechanism remains elusive. Hence, this model expands the current understanding of how acupoint sensitization enhances the acupuncture effect. Rather than acting as passive targets, sensitized acupoints are dynamically shaped by silent nociceptor activation, which transforms them into functionally excitable zones. Whether this priming involves sensory neuronal coupling, axon reflexes, or neuroimmune interactions remains to be explored in future studies [ 14 , 49 – 51 ] . Conclusions In summary, we identify CHRNA3⁺ silent nociceptors as essential modulators in shaping acupoint interface and transducing acupuncture signal. Their colitis-driven conversion to a mechanosensitive state primes the sensory interface, enabling the amplification of both pathological input and therapeutic signals. This reconfiguration positions CHRNA3⁺ MINs as a critical functional link connecting visceral disease-induced referral somatic area, acupoint, and acupuncture efficacy. Abbreviations AUC Area Under the Curve CGRP Calcitonin Gene-Related Peptide CHRNA3 Nicotinic Acetylcholine Receptor Subunit Alpha-3 CRD Colorectal Distension DAI Disease Activity Index DRG Dorsal Root Ganglion EA Electroacupuncture EB Evans Blue EMG Electromyography LFP Local Field Potential LTMR Low-Threshold Mechanoreceptor MIN Mechanoinsensitive Nociceptor NGF Nerve Growth Factor SP Substance P TDI Tissue Damage Index TNBS 2,4,6-Trinitrobenzenesulfonic Acid TrkA Tropomyosin Receptor Kinase A (NGF receptor) TRPV1 Transient Receptor Potential Vanilloid 1 VMR Visceromotor Reflex Declarations Funding This work was supported by Beijing Natural Science Foundation (7242247), the National Natural Science Foundation of China (No. 82230123, 81904309, 82174518), the Fundamental Research Funds for the Central public welfare research institutes (No. ZZ15-YQ-049, ZZ-YQ2023003), Scientific and technological innovation project of China Academy of Chinese Medical Sciences (CIZJS2025017). Author Contribution Study conception and design: X.C., W.X., X. G., B. Z.Conduct of experiments: W.X., D. Z., Y. T., Y. W., Z. W.Data analysis: X.C., W.X. D. Z., Y. T.Drafting of the manuscript: X.C., W.X., H. X.Editing and revision of the manuscript: X.C., X. G., B. Z.Approval of final version of the manuscript: all authors. Acknowledgement We are appreciating Zihan Xu, Yuetong Yin, and Yuting Lin from Beijing University of Chinese Medicine for their effort on the experiments. We are grateful to Professor Yun Guan and Drs. Qian Xu, Jing Liu and Qin Zheng from Johns Hopkins University School of Medicine for their constructive insights and discussion of data. Data Availability The data will be made available on reasonable request. References Kagitani F, Uchida S, Hotta H, Aikawa Y. Manual acupuncture needle stimulation of the rat hindlimb activates groups I, II, III and IV single afferent nerve fibers in the dorsal spinal roots. Jpn J Physiol. 2005;55(3):149–55. Lou XF, Jiang SH. 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Comparison of tissue damage index (TDI) scores from the histopathologic images among groups. ***p＜0.001, compared to saline; ###p＜0.01, compared to Model, n=6 per group. One-way ANOVA with the Bonferroni test. C. The scores of disease activity index (DAI) of rats in the three groups were compared. ***p＜0.001, compared to saline, n=6 per group. One-way ANOVA with the Bonferroni test. S2.jpg Supplementary Figure 2. Colitis increases NGF expression in colon (A), DRG (B), and BL25 skin (C). *P＜0.05, compared to Saline, n=3 per group; independent t-test. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 28 Jan, 2026 Reviews received at journal 23 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviews received at journal 03 Jan, 2026 Reviewers agreed at journal 03 Jan, 2026 Reviewers invited by journal 29 Dec, 2025 Submission checks completed at journal 29 Dec, 2025 First submitted to journal 04 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":366706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCHNRA3+ labeledmechanoinsensitive nociceptors (MINs) innervate peripheral tissue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAa-d.\u003c/strong\u003eImmunostaining of tissue sections showing CHRNA3⁺sensory fibers and neurons (red, white arrows) in the skin (a), colon (b), L6 DRG (c), and spinal cord (d). Magnified views of dashed white boxes are shown in (a1–a3, b1–b3, c1–c3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003eNeurochemical profiling of CHRNA3⁺nociceptors (red) by co-staining with A-nociceptor markers NF200 (a), TrkB (b) and C-nociceptor markers peripherin (c), non-peptidergic IB4 (d), and peptidergic CGRP (e) in rat DRG. Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003eProportion of co-localized neurons within the total CHRNA3+ population.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/c21c2e5202dc3356c5aab730.jpg"},{"id":97674330,"identity":"cf8424c3-dbfd-42ea-8073-a4b75d112171","added_by":"auto","created_at":"2025-12-08 09:42:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":452002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlockadeof CHRNA3+ nociceptor activation attenuated colitis-induced acupoint sensitization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eRepresentative images showing plasma extravasation (PE) points in the back 7 days after colitis induction. The extravasation of blue plasma proteins was validated using an in vivo imaging system (white arrow).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eQuantification of PE points. **p＜0.01vs. saline；##p＜0.01 vs. Model, n=6 per group. One-way ANOVA with Bonferroni test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Mapping of overlapped PE points (a) coinciding with classical acupoint locations (b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eSchematic of scAAV2/9-hSyn-EGFP and scAAV2/9-hSyn-mCherry injections into the colon and BL25, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Representative images showing co-labeling of GFP⁺colon-related neurons and mCherry⁺BL25-related neurons with CHRNA3 (magenta) and NeuN (cyan) in L6 DRG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eQuantification of triple-labeled neurons (GFP⁺/mCherry⁺/CHRNA3⁺) among NeuN⁺ neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u003c/strong\u003e Schematic diagram of mechanical threshold testing at the lumbosacral region.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.\u003c/strong\u003e Mechanical thresholds at BL25 before and 1, 3, 7 days after TNBS modeling. n=7 for saline, 8 for Model and Model+U0126 group. *P＜0.05, Model vs. Saline; ###P＜0.001 Model vs. Model+U0126. Two-way repeated measures ANOVA with Bonferroni post hoc test.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/fb6569f5b392d9a719bba37b.jpg"},{"id":97658933,"identity":"f05896ab-539d-4e89-a48f-9a9eb5957ca1","added_by":"auto","created_at":"2025-12-08 07:34:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":488421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColitis induces CHRNA3⁺MIN activation via ERK1/2 signaling in L6 DRG.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Schematic illustrating intracellular signaling pathways for CHRNA3⁺MIN activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eRepresentative in situ hybridization images of L6 DRG co-stained for \u003cem\u003echrna3\u003c/em\u003e (green), \u003cem\u003ePiezo2\u003c/em\u003e(magenta), and TrkA (red). Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eQuantification of triple-labeled (\u003cem\u003echrna3\u003c/em\u003e⁺/TrkA⁺/\u003cem\u003ePiezo2\u003c/em\u003e⁺) neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Representative immunostaining images of L6 DRG from Saline (a), Model (b), and Model+U0126 (c) groups stained for CHRNA3 (red) and pERK (green). higher magnifications shown in (a1–a3, b1–b3, c1–c3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eQuantification of CHRNA3⁺/pERK⁺ neurons among CHRNA3⁺ neurons.**p＜0.01vs. Saline；##p＜0.01 vs. Model, n=3 per group. One-way ANOVA with the Bonferroni test was used.\u003c/p\u003e\n\u003cp\u003eF. Representative immunoblotting images and quantification of protein levels of ERK1/2 and pERK1/2 in DRG of the Saline (a), Model (b), and Model+U0126 group. \u0026nbsp;*p＜0.05 vs. Saline；#p＜0.05 vs. Model, n=4 per group. One-way ANOVA with the Bonferroni test was used.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/253d894c4942cec323e94167.jpg"},{"id":97674307,"identity":"71083e18-0cf2-4f0c-a24e-bf5da4ea24fe","added_by":"auto","created_at":"2025-12-08 09:42:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":414744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColitisactivates TrkA⁺and PIEZO2⁺neurons in L6 DRG.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative images of TrkA⁺ (red) and pERK⁺(green) neurons in Saline (a), Model (b), and Model+U0126 (c) groups; magnified views in (a1–a3, b1–b3, c1–c3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Quantification of TrkA⁺/pERK⁺neurons among total TrkA⁺ neurons. **p＜0.01, vs. Saline；###p＜0.001 vs. Model, n=3 per group. One-way ANOVA with the Bonferroni test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Representative images of PIEZO2⁺ (red) and pERK⁺(green) neurons; magnified views in (a1–a3, b1–b3, c1–c3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Quantification of PIEZO2⁺/pERK⁺neurons in total PIEZO2+ neurons. *p＜0.05, vs. Saline；#p＜0.05 vs. Model, n=3 per group. One-way ANOVA with the Bonferroni test.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/8562f7f1cab430f05fe65ea6.jpg"},{"id":97674650,"identity":"8916bacc-cb46-4ebf-8872-f32797c7a137","added_by":"auto","created_at":"2025-12-08 09:43:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":213895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemogeneticinhibition of CHRNA3⁺MINs mitigates colitis-induced mechanical hypersensitivity at BL25.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003eSchematic of intracolonic injection of AAV virus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003eRepresentative images of ZsGreen-labeled neurons co-stained with CHRNA3 (red) and DAPI; magnified in (b1–b3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003eQuantification of ZsGreen⁺/CHRNA3⁺neurons among CHRNA3⁺ neurons. N=3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eTimeline of AAV-DREADD intervention and behavior testing. Chemogenetic inhibitory AAV-chrna3-hM4Di-ZsGreen (AAV-hM4Di) and control virus AAV-chrna3-ZsGreen (AAV-ZsGreen) were intracolonic injected in rats. Colitis modeling was conducted 21 days after virus injection to allow for sufficient transfection. Behavior tests were conducted before and 7 days after modeling, as well as the CNO administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Mechanical thresholds at BL25 pre- and post-TNBS modeling and pre- and post-CNO administration. N=8 per group. **P＜0.01, compared to baseline; ##P＜0.01, compared to pre-CNO; \u0026amp;\u0026amp; P＜0.01, compared to pre-CNO, compared to AAV-ZsGreen. Two-way repeated measure ANOVA with Bonferroni post hoc test.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/d8bd759fca581bf7d1d78daf.jpg"},{"id":97673086,"identity":"78d490fe-af51-40e9-8436-053db2d079ab","added_by":"auto","created_at":"2025-12-08 09:39:25","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":119123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemogeneticsilencing of CHRNA3+ MIN attenuates EA at BL25-induced analgesia on visceral hyperalgesia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eExperimental schematic of CRD-evoked VMR and EA protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003eRepresentative traces of CRD-evoked VMR EMG upon EA stimulation (BL 25, 1 mA, 2 Hz, 10 min), Pre- and Post-CNO administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003eFold change of area under the curve (normalized to baseline) of CRD-evoked VMR EMG before and post-EA intervention. n=6. ***P＜0.001, compared to Baseline; #P＜0.05, ##P＜0.01, compared to Pre-CNO. Two-way repeated measure ANOVA with Bonferroni post hoc test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eArea under the curve shows the calculated\u003cstrong\u003e \u003c/strong\u003echange of the CRD-evoked EMG after EA intervention pre and post CNO administration. Paired t-test.\u003cstrong\u003e \u003c/strong\u003e*P＜0.05, compared to Pre-CNO.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/d477b3ec9e33ccaa04f96428.jpg"},{"id":97658951,"identity":"3091d855-5704-4db3-89a8-43c51a1bc8d8","added_by":"auto","created_at":"2025-12-08 07:34:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":154916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of CHRNA3+ MIN potentiates EA-induced inhibition of spinal C-local field potentials (C-LFPs) in colitis rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eSchematic diagram of in vivo spinal LFP recording setup.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003eRepresentative trace for sciatic nerve stimulation-evoked LFP to A-fiber inputs (A-LFP) and C-fiber inputs (C-LFP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eRepresentative traces of evoked LFP in sham and colitis group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e. The areas under C-LFP curves of sham and colitis groups were plotted. N=8 per group. ***P\u0026lt;0.001, vs. Sham. Independent t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e. Experimental protocol for EA modulation of C-LFPs pre- and post-CNO in AAV-Chrna3-hM4Di-ZsGreen rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003eRepresentative trace of LFP before and after EA (BL 25, 1 mA, 2 Hz, 10 min) pre- and post-CNO administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u003c/strong\u003e. The AUC of C-LFP during each 5-minute period after EA were averaged for analysis. N=8. *P＜0.05, compared to baseline (time 0); #P＜0.05, compared to Pre-CNO; Two-way repeated measure ANOVA with Bonferroni post hoc test.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/e44c09d6ffcc534f91452c79.jpg"},{"id":97674824,"identity":"4e3e0f70-c368-4426-854d-3c0ce989e6b0","added_by":"auto","created_at":"2025-12-08 09:44:22","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":121234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematicdiagram summarizing how CHRNA3⁺MINs prime acupoint responsiveness and enhance acupuncture efficacy in colitis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eColitis leads to sensitization of acupoint BL25 (a) via axonal bifurcation of DRG neurons. Inflammatory components such NGF activate CHRNA3+ neurons, a subpopulation of C-nociceptors, through the TrkA/pERK/PIEZO2 signaling cascade (b). This converts CHRNA3+ neurons from mechanoinsensitive (silent) to mechanosensitive (awakened) state, contributing to colitis-induced mechanical hypersensitivity at BL25.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Awakened CHRNA3⁺ MINs primed the sensory interface of BL25 and mediated sensitization. This heightened responsiveness enhances the acupoint's reaction to EA stimulation, resulting in greater EA-induced analgesia for colitis-related visceral pain.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/82010ce17f98e3fe352a96bb.jpg"},{"id":97678810,"identity":"16b6f3e1-cb2e-4171-bac0-0e0e383745fd","added_by":"auto","created_at":"2025-12-08 09:56:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3561017,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/d572346e-520a-414f-b0d7-e56e1b61e102.pdf"},{"id":97658932,"identity":"f1468e5d-7913-42eb-a4d8-bfbaf59af2d6","added_by":"auto","created_at":"2025-12-08 07:34:30","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":137161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1 Blockade of CHRNA3+ nociceptor ameliorated TNBS-induced colitis, related to Figure2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eRepresentative histopathologic images of colon tissue 7 days after modeling for each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eComparison of tissue damage index (TDI) scores from the histopathologic images among groups. ***p＜0.001, compared to saline; ###p＜0.01, compared to Model, n=6 per group. One-way ANOVA with the Bonferroni test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eThe scores of disease activity index (DAI) of rats in the three groups were compared. ***p＜0.001, compared to saline, n=6 per group. One-way ANOVA with the Bonferroni test.\u003c/p\u003e","description":"","filename":"S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/08fd889981eb5a3d4616e806.jpg"},{"id":97674149,"identity":"45e65a55-8503-498d-841f-1355aaa71628","added_by":"auto","created_at":"2025-12-08 09:42:30","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":147351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Colitis increases NGF expression in colon (A), DRG (B), and BL25 skin (C). \u003c/strong\u003e*P＜0.05, compared to Saline, n=3 per group; independent t-test.\u003c/p\u003e","description":"","filename":"S2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6684432/v1/f01cd51fc76f817481ac4c0c.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"CHRNA3⁺ Nociceptors Prime the Cutaneous Sensory Interface to Enhance Electroacupuncture Analgesia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcupuncture has been practiced for millennia, yet the anatomical basis of acupoints remains elusive. Numerous studies have compared acupoints to adjacent non-acupoint regions, reporting subtle differences in sensory nerves\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, blood vessels, lymphatic vessels\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, connective tissues\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, tissue space areas\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, and mast cells\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. The absence of a strikingly anatomical signature raises a critical question: are acupoints fixed landmarks or dynamic, state-dependent sensory interfaces? Two longstanding challenges in acupuncture research compound this question. First, sham or \u0026ldquo;non-acupoint\u0026rdquo; controls often elicit effects comparable to true acupoints, undermining their validity. Second, the \u0026ldquo;wide-pan-acupoint\u0026rdquo; phenomenon, therapeutic benefit from needling outside traditional points, suggests that acupoint efficacy depends more on the local sensory state than on precise location. Together, these observations argue for reframing acupoints as plastic sensory interfaces, reshaped by disease, particularly visceral diseases, rather than immutable anatomical sites. Clinically, needling hypersensitive regions, such as Ashi and trigger points, consistently performs promising therapeutic outcomes, highlighting the potential role of sensory plasticity in acupoint function.\u003c/p\u003e\u003cp\u003eAcupoint sensitization is a recently characterized functional state in which somatic regions corresponding to acupoints exhibit sensory abnormalities under pathological conditions such as visceral disease. This state is defined by two key features: sensory hypersensitivity (e.g., mechanical allodynia) and heightened responsiveness to therapeutic stimuli like acupuncture\u003csup\u003e[\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Although these sensitized areas frequently overlap with regions of visceral referred pain\u003csup\u003e[\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, the core concept of acupoint sensitization emphasizes the resulting sensory abnormality and functional plasticity, which optimizes these sites for receiving and transducing therapeutic stimuli. Neurogenic inflammation is a key pathological driver of this sensitized state. It is initiated when activated sensory terminals release neuropeptides, stimulating local non-neuronal cells like mast cells and macrophages. This interaction establishes a self-amplifying feedback loop that primes the acupoint, enhancing its responsiveness to acupuncture. However, the precise mechanism through which visceral disease engages this neuroinflammatory cascade to modulate the acupoint's sensory interface remains undetermined.Peripheral sensory neurons, commonly referred to as nociceptors, emerging as critical players for acupuncture manipulation. Typically, C-nociceptors, comprised of peptidergic and non-peptidergic subtypes, have testified the essential role in the onset of acupoint sensitization\u003csup\u003e[\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Among these, silent nociceptors, also referred to as mechanoinsensitive nociceptors (MINs), have been implicated in the initiation of pain onset and exacerbation \u003csup\u003e[\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, and itch\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. These neurons, innervate visceral organs and somatic tissues\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, are typically unresponsive to mechanical stimuli under normal conditions but become sensitized and mechanosensitive in the presence of inflammatory mediators, such as nerve growth factor (NGF). Recent study has identified the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3), a subunit of the nicotinic acetylcholine receptor, as a specific molecular marker of MINs in mice\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. CHRNA3 neuron densely innervates somatic tissues and viscera and accounts for 40% of the peptidergic C-nociceptors, contributes to secondary mechanical hyperalgesia development\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Electrophysiological evidence reveals the \u0026ldquo;awaken\u0026rdquo; features of CHRNA3\u003csup\u003e+\u003c/sup\u003e neurons, which convert from mechano-insensitive to mechano-responsive following NFG through TrkA/pERK/PIEZO2 signaling pathway. Given this mechano-responsiveness similarity, we propose that CHRNA3\u003csup\u003e+\u003c/sup\u003e MIN mediated both the mechanical allodynia of sensitized acupoints and the amplification of sensory input during acupuncture.\u003c/p\u003e\u003cp\u003eIn this study, we identify CHRNA3⁺ neurons as a distinct subset of C-fiber nociceptors in rats, co-expressing with both peptidergic and non-peptidergic nociceptors. These neurons innervate visceral organs and homo spinal segmental somatic tissues, including the colon and the BL25 acupoint region, via axonal bifurcation, providing a structural substrate for viscerosomatic integration. Using a colitis-induced acupoint sensitization model, we demonstrate that CHRNA3⁺ MINs are activated by colonic inflammation and contribute to colitis-induced referred mechanical hypersensitivity at the BL25 acupoint, a classical site used in acupuncture for gut-related dysfunctions. Chemogenetic inhibition of CHRNA3⁺ MINs alleviated acupoint hyperalgesia and significantly reduced the efficacy of electroacupuncture (EA) in both behavioral and spinal neuronal readouts. These findings suggest that CHRNA3⁺ MINs not only mediate pathological sensitization but also act to prime and reshape the sensory interface of the acupoint, rendering it more responsive to therapeutic stimulation. This study presents a novel neurobiological mechanism through which silent nociceptors contribute to both the sensitization and functional plasticity of acupoints. Our findings bridge somatic sensory interface reshaping caused by visceral disease with acupoint and acupuncture effect, offering a mechanistic basis for understanding how nociceptor-mediated sensory interface prime acupoint function enhancing its modulatory effect.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eMale Sprague-Dawley rats (220\u0026thinsp;\u0026plusmn;\u0026thinsp;10 g) were purchased from Charles River (Beijing; license: SCXK-[Beijing]-2021-0011) and housed at the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (CACMS). Rats were maintained under standard laboratory conditions (12 h light/dark cycle, 24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, 60%\u0026ndash;70% humidity) and allowed to acclimate for one week before experimentation. All procedures were approved by the Institutional Animal Care and Use Committee of CACMS (Ethics No. Y2022-03-10-06).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTNBS-Induced Colitis Model\u003c/h3\u003e\n\u003cp\u003eColitis was induced as previously described\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. After a 24-hour fast, rats were anesthetized with 2% isoflurane, and TNBS (100 mg/kg in 50% ethanol, 2:1 ratio, Sigma-Aldrich) was administered intracolonically via a 30-gauge catheter inserted 6\u0026ndash;7 cm from the anus. Rats were held vertically for 5 min post-injection to ensure retention. Control rats received equal volumes of saline or 50% ethanol. Animals were monitored daily for body weight, general condition, and signs of colitis.\u003c/p\u003e\n\u003ch3\u003eDisease Activity Index (DAI) and Tissue Damage Index (TDI)\u003c/h3\u003e\n\u003cp\u003eColitis severity was assessed using the DAI and TDI scoring systems\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. DAI was calculated as the average of three parameters: weight loss (0\u0026ndash;4), stool consistency (0\u0026thinsp;=\u0026thinsp;normal, 2\u0026thinsp;=\u0026thinsp;loose, 4\u0026thinsp;=\u0026thinsp;diarrhea), and rectal bleeding (0\u0026thinsp;=\u0026thinsp;none, 2\u0026thinsp;=\u0026thinsp;occult, 4\u0026thinsp;=\u0026thinsp;gross). TDI was assessed on H\u0026amp;E-stained colon sections by two blinded pathologists, evaluating inflammatory infiltration, crypt loss, ulceration, and mucosal damage. Images were captured using a microscope (BX53, Olympus, Tokyo, Japan) and evaluated with Image J software (US National Institutes of Health, Bethesda, MD).\u003c/p\u003e\n\u003ch3\u003eEvans Blue Plasma Extravasation\u003c/h3\u003e\n\u003cp\u003eSkin plasma protein extravasation following Evans Blue (EB) administration in colitis rats was conducted as previously described\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Briefly, rats were anesthetized with 2% isoflurane at 7 days after colitis operation, and Evans Blue (50 mg/kg in saline, Sigma) was injected into the lateral tail vein. After 30\u0026ndash;60 min, PE points were mapped and counted. Hair was removed from the dorsal and hindlimb regions 24 h prior using depilatory cream (CP-8000, Codos, China).\u003c/p\u003e\n\u003ch3\u003eMechanical Sensitivity Testing\u003c/h3\u003e\n\u003cp\u003eMechanical thresholds at BL25 were measured using an electronic von Frey apparatus (ALMEMO 2450, AHLBORN) as previously described\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Rats were acclimated to a restraining pocket for 2 days and habituated for 30 min before each test. Measurements were taken on days 1, 3, and 7, and the mean of three trials was used.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAAV virus injection\u003c/h2\u003e\u003cp\u003eTo investigate whether dorsal root ganglion (DRG) neurons innervate both the colon and the BL25 acupoint region via axonal bifurcation, scAAV2/9-hSyn-EGFP (2.0 \u0026times; 10\u0026sup1;\u0026sup2; VG/mL, PT-2315, Shumi) and scAAV2/9-hSyn-mCherry (2.0 \u0026times; 10\u0026sup1;\u0026sup2; VG/mL, PT-3975, Shumi) were separately injected into the colonic wall and BL25 region. For intracolonic injections, a 1 cm midline abdominal incision was made at the rat under isoflurane anesthesia, and 10 \u0026micro;L of EGFP virus was injected between the muscularis externa and serosa at 5\u0026ndash;6 evenly spaced sites (~\u0026thinsp;2.5 cm total length) using a microsyringe under microscopic guidance. This multi-site, low-volume-per-site strategy was designed to maximize coverage while minimizing local tissue stress. For intracutaneous injections at BL25, a total of 10 \u0026micro;L of mCherry virus was delivered across 5\u0026ndash;8 evenly distributed points in the bilateral BL25 region. Ten days after injection, L6\u0026ndash;S1 DRGs were harvested, sectioned, and processed for colocalization analysis.\u003c/p\u003e\u003cp\u003eTo selectively inhibit colonic CHRNA3⁺ neurons, rAAV-Chrna3-ZsGreen (2.6 \u0026times; 10\u0026sup1;\u0026sup2; VG/mL) and rAAV-Chrna3-hM4D(Gi)-ZsGreen (Braincase) were injected into the colonic wall of the rat. A 1 cm incision was made under isoflurane anesthesia, and 10 \u0026micro;L virus was injected between the muscularis externa and serosa across 5\u0026ndash;6 sites (~\u0026thinsp;2.5 cm span). Colitis modeling was conducted 21 days after AAV injection to allow for sufficient transgene expression. Behavioral and electrophysiological experiments were conducted 7 days after colitis modeling (28 days after virus injection). The timeline was consistent for all chemogenetic experiments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunofluorescence and In Situ Hybridization\u003c/h3\u003e\n\u003cp\u003e Rats were perfused with PBS followed by 4% paraformaldehyde. DRG, skin, colon, and spinal tissues were harvested, cryoprotected in 30% sucrose, embedded in OCT, and cryosectioned at 20 \u0026micro;m. Sections were blocked with 10% serum and incubated overnight at 4\u0026deg;C with primary antibodies, followed by fluorescent secondary antibodies and DAPI. Confocal imaging was performed using an FV1000 microscope (Olympus, Tokyo, Japan). The following primary antibodies were used: CHRNA3, (Invitrogen, PA5-77501, 1:200), PIEZO2 (Novus, NBP2-58161, 1:50), TrkA (R\u0026amp;D, AF1056, 1:200), TrkA (NOVUS, AF1494, 1:200), p-ERK (Santa Cruz, sc-7383, 1:200), NeuN (Sigma\u003c/p\u003e\u003cp\u003eRNAscope Multiplex Fluorescent Reagent Kit V2 (Cat. No. 323110) was used to perform in situ hybridization with \u003cem\u003echrna3\u003c/em\u003e (527051-C1, ACD) and \u003cem\u003ePIEZO2\u003c/em\u003e (549741-C3, ACD) probes according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003eWestern blot assay\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot assay\u003c/div\u003e\u003cp\u003eSeven days after colitis modeling, the bilateral lumbar L6-S1 DRG was collected and homogenized in ice-cold RIPA lysis buffer (89900, Thermo Scientific) with Halt Protease Phosphatase Inhibitor (5872s, CST, 1:500). Protein concentrations were measured with a Pierce BCA Protein Assay Kit (23225, Thermo Scientific). Equal amounts of the proteins sample were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes. Proteins samples were then incubated with antibodies for rabbit anti-ERK1/2 (A4782, Abclonal, 1:2000), rabbit anti-pERK1/2 (AP0472, Abclonal, 1:1000), rabbit anti-NGF (Abcam, Abclonal, ab52918, 1:2000), and β-actin (XAb-A, X-blot) overnight at 4℃, followed by incubation with the relative secondary antibodies. The interested bands were visualized using an ECL kit (170\u0026ndash;5060, BIO-RAD) and imaged with iBright1500 (Invitrogen). The β-actin served as the loading control and intensity of the selected bands was analyzed using Image J.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eU0126 Treatment\u003c/h2\u003e\u003cp\u003eU0126 (5 mg/kg/day, i.p.) was administered to rat for 7 days after colitis induction. Stock solution (50 mg/mL in DMSO) was diluted to 5 mg/mL in 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eElectroacupuncture (EA)\u003c/h2\u003e\u003cp\u003eRats were placed in a prone position on a heating pad and anesthetized with inhalation of 2% isoflurane. The bilateral BL25 acupoints are located at the depression lateral to the lower border of the spinous process of the fourth lumbar vertebra. The needles (0.25\u0026times;25 mm, Suzhou Medical Appliance Factory, Suzhou, China) were inserted into the bilateral BL25 point at a depth of ~\u0026thinsp;3 mm. EA were delivered using a stimulator (STG4000, Warner Instruments, U.S.A) with the following parameters\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e: 2 Hz frequency, 1 mA intensity, for a duration of 10 min.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eVisceromotor Reflex (VMR) Recording\u003c/h2\u003e\u003cp\u003eVisceromotor response (VMR) was recorded as an indicator of Visceral hyperalgesia (VH) by colorectal distension (CRD) which was aroused by using a custom-made balloon\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. After mice were anesthetized with 2% isoflurane, a lubricated polyethylene balloon (1cm diameter) was inserted transanally into the colorectum approximately 0.5-1 cm from the anal verge. The external portion was secured to the tail with tape. Colorectal distension was produced by rapidly inflating the balloon to a constant pressure of 60 mmHg. The pressure was maintained for 20 seconds and allowed 5 min for recovery between each inflation. EMG spikes were recorded by the PowerLab Data Recording \u0026amp; Analysis System (ADIstruments Pty Ltd).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSpinal LFP Recording\u003c/h2\u003e\u003cp\u003eThe experimental setup for in vivo spinal LFP recording was similar to our previous studies.5\u0026ndash;7 Briefly, the rats were anesthetized with urethane (1.2 g/kg, i.p.). The lumbar spinal cord was exposed in anesthetized rats and the dura mater was partially removed at the recording segments (L6-S1). The paralyn-coated tungsten microelectrode (3 mΩ, Frederick Haer Company, Brunswick, ME, USA) was inserted into the superficial dorsal horn (200\u0026ndash;500 \u0026micro;m below the surface) at L6 spinal segment. The left sciatic nerve was exposed and placed on the hook electrode to deliver the test stimulus (5 mA, 0.2 msec, 1 test/1 min), evoking the spinal LFP. A real-time, computer-based data acquisition and processing system (CED Spike 2, Cambridge, UK) was used to collect analog data. Raw data was collected at a sampling rate of 1000 Hz. The data stream was amplified and then filtered (0.1\u0026ndash;100 Hz, model DAM80; World Precision Instruments, Sarasota, FL, USA), and artifacts of stimulation were removed online by a notch filter (IIR filters of the CED 1401 data-acquisition system).\u003c/p\u003e\u003cp\u003eLFP was examined before EA (baseline, 10 min) and at 0\u0026ndash;30 min after EA application. Based on the conduction velocity, LFPs that correspond to activated A- and C-fibers can be distinguished. In comparison to the large A-LFP, the C-LFP exhibits a longer latency (90\u0026ndash;130 msec) and smaller amplitude. Offline modulus measurements of Spike2 software were used to analyze the area under curves (AUC) of C-LFP. A comparison was made between pre- and post-EA conditions within and between the groups after normalizing LFP to pre-EA baseline values in each animal.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eSample sizes (n\u0026thinsp;=\u0026thinsp;6\u0026ndash;8 per group) were determined based on established standards in the field for similar models and endpoints, which consistently yield large effect sizes. This approach aligns with the ethical goal of minimizing animal use while ensuring robust detection of biological effects. The GraphPad Prism (version 8.0, GraphPad Software, San Diego, CA, USA) software was used for statistical analysis. The normality of data was checked using the Shapiro‒Wilk test. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. A paired or independent t-test was used to analyze data within two groups. One-way or two-way ANOVA with post hoc Bonferroni test was utilized for group comparison. Significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCHRNA3⁺ nociceptors innervate peripheral tissues and prime acupoints for colitis-induced sensitization\u003c/h2\u003e\u003cp\u003eConcerning previous studies of CHRNA3 neurons in pain conducted in mice, we herein examined the innervation patterns of CHRNA3\u0026thinsp;+\u0026thinsp;mechanoinsensitive nociceptors (MINs) in rats for the first time. Using immunofluorescence, we observed that CHRNA3⁺ MINs innervate the lumbar skin, colon, L6 dorsal root ganglion (DRG), and lamina I of the L6\u0026ndash;S1 spinal dorsal horn (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), indicating their widespread peripheral and central connectivity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine the nociceptor subtype identity of CHRNA3⁺ neurons, we performed co-staining with markers for A-fiber neurons (NF200, TrkB), C-fiber markers (Peripherin), and subtype indicators for peptidergic (CGRP⁺) and non-peptidergic (IB4⁺) populations. CHRNA3⁺ neurons showed minimal overlap with A-fiber markers (NF200: 7.94%; TrkB: 8.37%) but significant co-expression with C-fiber markers (Peripherin: 50.29%), IB4 (72.69%), and CGRP (44.55%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;C). These data confirm that CHRNA3 is predominantly expressed in C-fiber nociceptors, convering both peptidergic and non-peptidergic subtypes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCHRNA3⁺ MINs Mediate Colitis-Induced Acupoint Sensitization\u003c/h2\u003e\u003cp\u003eTo assess the functional involvement of CHRNA3⁺ MINs in acupoint sensitization, we employed a TNBS-induced colitis model known to produce referred somatic hypersensitivity\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Evans blue (EB) dye was injected intravenously to visualize sites of neurogenic inflammation, marked by plasma extravasation (PE) points\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Colitis significantly increased the number of PE points in the abdominal and lumbar regions compared to saline controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C), and these overlapped with classical acupoints such as BL25 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-b), consistent with previous study about the sensitized acupoints under colitis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInhibition of CHRNA3⁺ MINs with U0126, a pERK inhibitor, significantly reduced the number of PE points. Given that mechanical hypersensitivity is a hallmark of sensitized acupoints, colitis-induced sensitized acupoint BL25 was selected and mechanical thresholds was assesedusing electronic von Frey testing. Colitis significantly reduced mechanical thresholds at BL25, indicating mechanical allodynia, which was ameliorated by U0126 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u0026ndash;F). These findings suggest that activation of CHRNA3⁺ MINs is necessary for the onset of acupoint hypersensitivity and mechanical sensitization during colitis.\u003c/p\u003e\u003cp\u003eTo investigate whether axonal bifurcation of CHRNA3⁺ neurons mediate the interaction between the colon and the BL25 acupoint region, scAAV2/9-hSyn-EGFP and scAAV2/9-hSyn-mCherry were injected separately into the colonic wall and the BL25 region, respectively. Co-staining with CHRNA3 and NeuN revealed that approximately 8.55% of total NeuN⁺ DRG neurons were triple-labeled with GFP, mCherry, and CHRNA3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;E). These findings indicate that a subset of CHRNA3⁺ neurons simultaneously innervate both the colon and the skin, providing a neuroanatomical basis for viscerosomatic crosstalk.\u003c/p\u003e\u003cp\u003eAdditionally, we examined whether CHRNA3⁺ MINs contribute to tissue injury during colitis. TNBS significantly increased the colonic tissue damage index (TDI) and disease activity index (DAI) (Supplementary Fig.\u0026nbsp;1). Interestingly, U0126 reduced TDI scores but not DAI, implying that CHRNA3⁺ MINs may selectively modulate local tissue injury responses without altering overall disease burden.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eCHRNA3⁺ MIN Activation Is Driven by TrkA/pERK/PIEZO2 Signaling\u003c/h2\u003e\u003cp\u003ePrevious work in mice identified TrkA/pERK/PIEZO2 signaling as a key pathway underlying silent nociceptor activation\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Using in situ hybridization and immunofluorescence in rat L6 DRG, we confirmed that 47.33% of CHRNA3⁺ neurons co-expressed both TrkA and PIEZO2, while 25.03% of TrkA⁺ and 45.67% of PIEZO2⁺ neurons were triple-labeled (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;C; Supplementary Figure S2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next investigated whether colitis activates CHRNA3⁺ MINs through this pathway. Immunostaining showed a marked increase in pERK⁺ CHRNA3⁺ neurons in the DRG of colitis rats compared to saline controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;E). U0126 treatment significantly reduced this activation. Similarly, pERK co-localization with TrkA⁺ and PIEZO2⁺ neurons increased after colitis and was suppressed by U0126 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). To determine whether NGF is a potential upstream driver\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, we assessed NGF expression in the colon, DRG, and BL25 skin. NGF was significantly upregulated in colon and DRG, as well as an elevation trend in skin following colitis (Supplementary Fig.\u0026nbsp;2), supporting a role for NGF\u0026ndash;TrkA\u0026ndash;pERK\u0026ndash;PIEZO2 signaling in CHRNA3⁺ MIN activation. These data strongly support a mechanism in which colitis activates CHRNA3⁺ MINs via NGF-dependent signaling, promoting their conversion from a mechanoinsensitive to a mechanoresponsive state\u0026mdash;thus priming the acupoint for heightened sensory input.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eChemogenetic Inhibition of CHRNA3⁺ MINs Alleviates Acupoint Hyperalgesia\u003c/h2\u003e\u003cp\u003eTo directly assess the contribution of CHRNA3⁺ MINs to mechanical hypersensitivity, we constructed an AAV vector expressing inhibitory DREADD (hM4Di) under the \u003cem\u003eChrna3\u003c/em\u003e promoter (AAV-Chrna3-hM4Di-ZsGreen) and confirmed that ~\u0026thinsp;91.67% of CHRNA3⁺ neurons expressed the reporter three weeks post-intracolonic injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C). CNO administration in AAV-hM4Di-transduced rats significantly increased mechanical thresholds at BL25 compared to pre-CNO and control virus (AAV-ZsGreen) animals, indicating attenuation of colitis-induced mechanical hypersensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;E). These results functionally confirm that CHRNA3⁺ MIN plays a role in impacted acupoint microenviroment, which then primed the interface and sustained the sensitized acupoint state for pathological mechanical responses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eCHRNA3⁺ MINs Facilitate Electroacupuncture-Induced Analgesia\u003c/h2\u003e\u003cp\u003eTo determine whether CHRNA3⁺ MINs enhance acupuncture signaling, we assessed the analgesic effects of electroacupuncture (EA) at BL25 on colorectal distension (CRD)-evoked visceral motor reflexes (VMRs)\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. EA significantly reduced the EMG area under the curve (AUC) during CRD in colitis rats, while CNO-induced silencing of CHRNA3⁺ neurons attenuated this inhibitory effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;C), suggesting that CHRNA3⁺ MINs facilitate EA-induced analgesia.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate central effects, we recorded spinal cord C-local field potentials (C-LFPs) in response to sciatic nerve stimulation. Colitis elevated C-LFP amplitudes compared to Sham, reflecting spinal sensitization\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. EA at BL25 suppressed C-LFPs at 5, 10, and 15 min, but this suppression was abolished by CHRNA3⁺ neuron silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF\u0026ndash;G). Together, these data determined that CHNRA3\u0026thinsp;+\u0026thinsp;MIN is involved in acupuncture\u0026rsquo;s signals transduction, suggesting CHRNA3⁺ MINs not only primes peripheral acupoints but also enables EA-driven inhibition of spinal nociceptive transmission.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIncreasing evidence suggests that acupoints are associated with disease-induced referred somatic pain, a phenomenon known as acupoint sensitization\u003csup\u003e[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. However, the neural basis of acupoint sensitization, and how it mediates and transmits acupuncture signals, remains poorly understood. In this study, we identified CHRNA3, a known marker of mechanoinsensitive nociceptors (MINs) in mice, as being predominantly expressed in C-fiber nociceptors in rats, with widespread innervation of peripheral tissues. We further demonstrated that activation of CHRNA3⁺ MINs contributes to colitis-induced acupoint sensitization and mechanical hypersensitivity at the BL25 acupoint via the TrkA/pERK/PIEZO2 signaling pathway. Notably, these neurons appear to prime the peripheral sensory field, enhancing the responsiveness of acupoints to both nociceptive and therapeutic stimuli, such as EA. At the same time, colitis-driven CHRNA3⁺ MIN activation reshapes the acupoint sensory interface, converting it into a hyperexcitable state that facilitates signal amplification. Accordingly, chemogenetic inhibition of CHRNA3⁺ MINs not only alleviated mechanical hyperalgesia at sensitized BL25 but also attenuated the analgesic effects of EA on colitis-related visceral hypersensitivity. These findings highlight a dual role for CHRNA3⁺ MINs in priming and reshaping acupoint function, thereby modulating both pathological sensitization and acupuncture-induced analgesia (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003eNociceptors Mediate Acupoint Sensitization\u003c/h2\u003e\u003cp\u003eThe concept of acupoint sensitization has gained attention in recent decades, supported by clinical and preclinical observations that many acupoints overlap with regions of referred somatic hypersensitivity associated with visceral disease or tissue injury. Sensory hypersensitivity and functional enhancement are hallmark features of this phenomenon. Notably, mechanical allodynia is considered the primary manifestation of acupoint sensitization\u003csup\u003e[\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, aligning with the classical concept of \"selecting the painful point as the acupuncture point\" described in the \u003cem\u003eJing Jin\u003c/em\u003e chapter of the \u003cem\u003eMiraculous Pivot\u003c/em\u003e. In this study, we confirmed that regions of plasma extravasation overlapped with colitis-related acupoints, supporting the pathological relevance of referred hypersensitivity\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, although both the colon and the BL25 acupoint are innervated by the same spinal segments (L6\u0026ndash;S1), we herein demonstrate that axonal bifurcation may serve as a potential neuroanatomical basis for colitis-induced acupoint sensitization.\u003c/p\u003e\u003cp\u003eWhile multiple studies have explored anatomical substrates contributing to acupoint function, including sensory nerves\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, blood vessels, lymphatic vessels\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, connective tissues\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, tissue space areas\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, and mast cells\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, no single structure clearly distinguishes acupoints from adjacent non-acupoint regions. This has led to a shift in understanding acupoints not as fixed structures, but as primed sensory interfaces at the somatic area, dynamically modulated by pathophysiological states and capable of integrating acupuncture inputs\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAcupoints contain a complex neuroimmune microenvironment involving nociceptors, immune cells, blood vessels, and autonomic terminals. Among these, nociceptors are thought to serve as primary transducers of acupuncture signals, and increasing evidence supports their essential role in acupoint sensitization and EA-induced neuromodulation\u003csup\u003e[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In a rat model of knee osteoarthritis, Zhang et al. found that sensitized ST35-associated DRG neurons displayed enhanced excitability and Ih-current density in C-fibers, but not Aδ-fibers\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Other studies have shown that acupoint sensitization is associated with C-fiber-mediated neurogenic inflammation, evidenced by elevated CGRP and SP levels in the skin\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Our findings extend this by demonstrating that CHRNA3⁺ MINs, despite only partial overlap with CGRP (\u0026sim;40%), play a critical role in initiating acupoint sensitization. Chemogenetic inhibition of CHRNA3⁺ neurons reduced colitis-induced plasma extravasation and mechanical allodynia, supporting their functional involvement. Recent studies reveal the immune cells and even the immune process were modulated by the nociceptor-driven neuropeptide such as CGRP and SP, which produces dual role in different pathological contexts\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Whether and how CHRNA3\u0026thinsp;+\u0026thinsp;MINalso engage with immune cells to shape acupoint sensory interface remains an open question for future studies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eSilent Nociceptor Activation Primes the Acupoint Sensory Interface\u003c/h2\u003e\u003cp\u003eC-fiber nociceptors comprise a diverse population with distinct molecular and functional properties. Single-cell RNA sequencing has revealed at least 18 transcriptomic clusters\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. These neurons can be broadly categorized into peptidergic (e.g., CGRP⁺, SP⁺) and non-peptidergic (e.g., IB4⁺) subtypes. CHRNA3 is predominantly co-expressed with Peripherin and colocalizes with both peptidergic and non-peptidergic C-nociceptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;C). Functionally, C-nociceptors are further distinguished by their responsiveness to thermal, chemical, or mechanical stimuli, which may shift under pathological conditions\u003csup\u003e[\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSilent nociceptors represent a unique subtype that are typically unresponsive to mechanical stimuli under normal conditions but acquire mechanosensitivity following inflammation or injury via the TrkA/pERK/PIEZO2 axis\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. This \"awakening\" process is well documented across species: approximately 15\u0026ndash;20% of cutaneous C-fibers in humans are silent nociceptors, with even higher proportions (30\u0026ndash;90%) reported in visceral and articular tissues in rodents\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. In a mouse model of colitis, the proportion of mechanosensitive C-fibers rose from 34% to 53%, while silent nociceptors decreased from 27% to 13%, implying their conversion to an active state. Recent work also highlights the role of MINs in secondary mechanical hypersensitivity in joint inflammation\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe Lechner group identified CHRNA3 as a reliable molecular marker for MINs in mice and showed that their activation is driven by NGF through the TrkA/pERK/PIEZO2 signaling cascade\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Subsequent studies of Lechner group identified TMEM100 as a key downstream molecular switch regulating silent nociceptor activation and mediate secondary hyperalgesia\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Based on these insights, we propose that CHRNA3⁺ MINs play a dual role in both the development of mechanical allodynia at sensitized acupoints and the amplification of acupuncture signals. Consistent with this model, our data confirmed co-expression of CHRNA3 with TrkA, pERK, and PIEZO2 in rat DRG neurons and demonstrated that CHRNA3⁺ MINs are activated by colitis. Despite observing only a trend toward increased NGF in the skin in this study, NGF levels in colon and DRG were significantly elevated under colitis condition. This suggests spatial heterogeneity in NGF regulation across sensory neurons, the primary site of pathology, and their referred peripheral terminals. Plus, we also observed that participation of NGF-TrkA/pERK/PIEZO2 signaling in acupoint sensitization, suggested by the increased co-expression portion of TrkA and PEIZO2 with pERK under TNBS and reversed upon U0126 administration. Chemogenetic silencing of these neurons abolished their mechanical conversion and reduced both acupoint sensitization and mechanical hypersensitivity, supporting their crucial role in priming the sensory interface. However, because we did not assess CHRNA3 expression in autonomic ganglia, including the pelvic ganglion, or in the nodose ganglion (which is critical for interoceptive signaling) during our chemogenetic viral experiments, the potential contributions of CHRNA3⁺ neurons in these ganglia remain unclear and warrant further investigation.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eCHRNA3⁺ MINs Amplify Acupuncture Signaling\u003c/h2\u003e\u003cp\u003eFunctionally, CHRNA3⁺ MIN activation appears to enhance the analgesic effects of acupuncture. Silencing CHRNA3⁺ neurons attenuated EA-induced inhibition of visceral pain responses and spinal neuronal hyperactivation. Previous studies have shown that C-nociceptors are essential for acupuncture effects: for example, TRPV1 knockout mice exhibit impaired EA-induced analgesia\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e,, and \u003cem\u003eProkr2\u003c/em\u003e sensory neurons mediate EA-driven anti-inflammatory responses\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. However, most prior work has focused on how somatic sensory input generates downstream effects. In contrast, our study reveals a novel mechanism whereby CHRNA3⁺ MINs prime the sensory interface, rendering acupoints more responsive to mechanical and EA stimulation. Increasing evidence demonstrated the superior therapeutic effect when targeting sensitized acupoints, such as the management of chronic musculoskeletal and non-musculoskeletal pain\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, bronchial asthma\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e, and allergic rhinitis\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e, but the mechanism remains elusive. Hence, this model expands the current understanding of how acupoint sensitization enhances the acupuncture effect. Rather than acting as passive targets, sensitized acupoints are dynamically shaped by silent nociceptor activation, which transforms them into functionally excitable zones. Whether this priming involves sensory neuronal coupling, axon reflexes, or neuroimmune interactions remains to be explored in future studies\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we identify CHRNA3⁺ silent nociceptors as essential modulators in shaping acupoint interface and transducing acupuncture signal. Their colitis-driven conversion to a mechanosensitive state primes the sensory interface, enabling the amplification of both pathological input and therapeutic signals. This reconfiguration positions CHRNA3⁺ MINs as a critical functional link connecting visceral disease-induced referral somatic area, acupoint, and acupuncture efficacy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAUC Area Under the Curve\u003c/p\u003e\u003cp\u003eCGRP Calcitonin Gene-Related Peptide\u003c/p\u003e\u003cp\u003eCHRNA3 Nicotinic Acetylcholine Receptor Subunit Alpha-3\u003c/p\u003e\u003cp\u003eCRD Colorectal Distension\u003c/p\u003e\u003cp\u003eDAI Disease Activity Index\u003c/p\u003e\u003cp\u003eDRG Dorsal Root Ganglion\u003c/p\u003e\u003cp\u003eEA Electroacupuncture\u003c/p\u003e\u003cp\u003eEB Evans Blue\u003c/p\u003e\u003cp\u003eEMG Electromyography\u003c/p\u003e\u003cp\u003eLFP Local Field Potential\u003c/p\u003e\u003cp\u003eLTMR Low-Threshold Mechanoreceptor\u003c/p\u003e\u003cp\u003eMIN Mechanoinsensitive Nociceptor\u003c/p\u003e\u003cp\u003eNGF Nerve Growth Factor\u003c/p\u003e\u003cp\u003eSP Substance P\u003c/p\u003e\u003cp\u003eTDI Tissue Damage Index\u003c/p\u003e\u003cp\u003eTNBS 2,4,6-Trinitrobenzenesulfonic Acid\u003c/p\u003e\u003cp\u003eTrkA Tropomyosin Receptor Kinase A (NGF receptor)\u003c/p\u003e\u003cp\u003eTRPV1 Transient Receptor Potential Vanilloid 1\u003c/p\u003e\u003cp\u003eVMR Visceromotor Reflex\u003c/p\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by Beijing Natural Science Foundation (7242247), the National Natural Science Foundation of China (No. 82230123, 81904309, 82174518), the Fundamental Research Funds for the Central public welfare research institutes (No. ZZ15-YQ-049, ZZ-YQ2023003), Scientific and technological innovation project of China Academy of Chinese Medical Sciences (CIZJS2025017).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eStudy conception and design: X.C., W.X., X. G., B. Z.Conduct of experiments: W.X., D. Z., Y. T., Y. W., Z. W.Data analysis: X.C., W.X. D. Z., Y. T.Drafting of the manuscript: X.C., W.X., H. X.Editing and revision of the manuscript: X.C., X. G., B. Z.Approval of final version of the manuscript: all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are appreciating Zihan Xu, Yuetong Yin, and Yuting Lin from Beijing University of Chinese Medicine for their effort on the experiments. We are grateful to Professor Yun Guan and Drs. Qian Xu, Jing Liu and Qin Zheng from Johns Hopkins University School of Medicine for their constructive insights and discussion of data.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data will be made available on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKagitani F, Uchida S, Hotta H, Aikawa Y. 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Front Immunol, 2021. 12.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Acupoint sensitization, Mechanoinsensitive nociceptors, Silent nociceptors, Electroacupuncture","lastPublishedDoi":"10.21203/rs.3.rs-6684432/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6684432/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eRecent evidence determined that acupoints frequently overlap with regions of referred somatic hypersensitivity induced by visceral disease, a phenomenon known as acupoint sensitization. This state is typically characterized by sensory hypersensitivity and functional enhancement, often accompanied by superior therapeutic outcomes following acupuncture. However, the neurobiological mechanisms that prime acupoints for enhanced responsiveness remain poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eUsing a rat model of TNBS intracolonic injection-induced colitis and relative acupoint sensitization, we investigated the role of CHRNA3⁺ mechanoinsensitive nociceptors (MINs), a subclass of silent C-fiber neurons, in modulating the acupoint sensory interface and electroacupuncture (EA) responsiveness. Behavior tests, neuroanatomical tracing, \u003cem\u003ein situ\u003c/em\u003e hybridization, pharmacological blockade, and chemogenetic silencing were employed to assess the involvement of CHRNA3⁺ MINs in the onset of acupoint sensitization. Colonic distension-evoked visceral motor reflex and spinal local field potential recording were utilized to evaluate the contribution of CHRNA3⁺ MINs in the acupuncture-induced analgesic effect.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe found that CHRNA3⁺ MINs are primarily C nociceptors co-expressing TrkA, pERK, and PIEZO2, and that they innervate both the colon and lumbar skin (BL25 acupoint region) via axonal bifurcation. Colitis significantly activated CHRNA3⁺ nociceptors via the NGF\u0026ndash;TrkA/pERK/PIEZO2 pathway, converting them from mechanoinsensitive to mechanosensitive. This activation correlated with increased plasma extravasation and mechanical allodynia at BL25. Pharmacological inhibition of CHRNA3⁺ MINs via pERK blockade or chemogenetic silencing reduced mechanical hypersensitivity of BL25 and attenuated the analgesic effects of EA on both visceral pain and spinal sensitization.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur findings reveal that CHRNA3⁺ silent nociceptors dynamically prime and reshape the sensory interface of the colitis-induced sensitized acupoint BL25, facilitating both pathological hypersensitivity and therapeutic responsiveness. This study establishes a mechanistic link between visceral dysfunction, acupoint functional plasticity, and EA-induced therapeutic neuromodulation.\u003c/p\u003e","manuscriptTitle":"CHRNA3⁺ Nociceptors Prime the Cutaneous Sensory Interface to Enhance Electroacupuncture Analgesia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 07:34:25","doi":"10.21203/rs.3.rs-6684432/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-29T03:04:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-23T14:31:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280907363299037378034023603735189647425","date":"2026-01-14T08:34:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-03T08:04:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164239414695151349156376870740321326454","date":"2026-01-03T06:24:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-30T04:49:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-30T04:24:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2025-12-04T09:08:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2beb931b-ef96-4ac6-93f7-e396b58bf33e","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T05:55:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 07:34:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6684432","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6684432","identity":"rs-6684432","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}