A total of 21 adult mice (22–25 g body weight) were used in this study, comprising 6 C57BL/6 wild-type mice, 2 TrpV1^𝐶𝑟𝑒 mice, 8 TrpV1^ChR2-eYFP mice (expressing ChR2-EYFP), and 5 TrpV1^NpHR-eYFP mice (expressing NpHR3.0-EYFP). The sex of the animals was not distinguished in the experimental design. C57BL/6 mice (22–25 g) were obtained from SPF Biotechnology Co., Ltd. (Beijing, China). The TrpV1^𝐶𝑟𝑒 mouse line (Stock No. 017769) was generously provided by Dr. Shenbin Liu’s laboratory at Fudan University. Optogenetic mouse lines, including ChR2 (Ai32; Stock No. 024109) and NpHR (Ai39; Stock No. 014539), were acquired from The Jackson Laboratory (Bar Harbor, ME, USA). Transgenic TrpV1^ChR2-eYFP and TrpV1^NpHR-eYFP mice (22–25 g) were generated and maintained by Shanghai Model Organisms Center, Inc. (Shanghai, China).

All animals were housed in standard polycarbonate cages (maximum 5 mice per cage) with ad libitum access to autoclaved water and standard rodent chow. The vivarium was maintained at 22 $\pm$ 1 °C, 50 $\pm$ 5% relative humidity, and a 12:12 h light-dark cycle (lights on at 07:00). All experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Experimental Animal Ethics Committee of the Chinese Medical Sciences (No. D2024-01-26-07). Procedures were performed under isoflurane anesthesia (induction: 4%, maintenance: 1.5–2% in 100% O₂), with body temperature maintained at 37 $\pm$ 0.5 °C using a feedback-controlled heating pad. Euthanasia was performed by isoflurane (5%) followed by cervical dislocation.

The fabricated polyethylene balloon catheters used here were described previously [13]. Catheter integrity was validated prior to experimentation via underwater pressure testing (1 mL syringe inflation) to confirm the balloon membrane integrity and leak-proof tubing connections. Following 4-h fasting (ad libitum water access), mice were anesthetized with 4% isoflurane in oxygen for induction and 2% isoflurane via nose cone for maintenance. The lubricated balloons were inserted 2 cm beyond the anal verge.

Following surgical preparation, a three-way stopcock was connected to a calibrated pressure transducer (Biopac Systems) pre-filled with distilled, deionized water. The system was meticulously purged of air bubbles to ensure signal fidelity, and the recording apparatus was then initialized and calibrated. This comprised a NeuroLog NL108A amplifier (Digitimer Ltd., Letchworth, UK), Micro1401 data acquisition interface (Cambridge Electronic Design, Cambridge, UK), and Spike2 software (v7.03, Cambridge Electronic Design, Cambridge, UK).

The stopcock configuration was adjusted to establish atmospheric pressure equilibration within the balloon system. Baseline calibration was performed to establish a 0 cmH₂O reference. A precise volume of distilled water (50–100 µL) was injected into the balloon using a calibrated microsyringe (Hamilton Company, Bonaduz, Switzerland). The spherical water-filled balloon, positioned within the colorectal lumen, transduced intraluminal pressure changes, which were transmitted hydrostatically through the polyethylene catheter to the pressure transducer. The analog pressure signal was amplified (NeuroLog NL108A) and digitized (Micro1401) for acquisition. Real-time signal visualization and recording were performed using Spike2 (v7.03, Cambridge Electronic Design Limited, British) with appropriate filtering.

Following stabilization of the colorectal motility patterns (typically 15–30 minutes post-surgical preparation), baseline recordings were acquired for 1 minute prior to stimulation. Each stimulation protocol was administered for precisely 1 minute, followed by a minimum 5-minute washout period to allow complete recovery of motility parameters to baseline levels, as confirmed by real-time monitoring of pressure traces. This inter-stimulus interval was maintained to prevent carryover effects between experimental conditions. Comparative waveform analysis between pre-stimulation baseline and stimulation periods was performed using two key parameters: Area Under Curve (AUC), and Contractile Amplitude. AUC, pressure-time integral, represents the total propulsive effort (cmH₂O$\cdot$s), while amplitude ($\mathrm{\Delta }$p = max–min) reflects instantaneous neuromuscular drive (cmH₂O).

The ST36 acupoint was localized according to standardized anatomical landmarks described in the Experimental Acupuncture reference text. It was identified as 3 mm lateral to the tibial crest at the knee joint level, corresponding to the classical anatomical position in murine models. Stimulation duration was maintained for 1 minute.

EA was administered using acupuncture needles connected to a Constant Current Isolated Stimulator (DS3; Digi-timer Ltd., Welwyn Garden City, UK). Stimulation parameters were set at 1 mA intensity, 10 Hz frequency, and 1 ms pulse width. The needle was inserted vertically to a depth of 5 mm at the ST36 acupoint. Manual Acupuncture (MA) was performed by a single trained practitioner to ensure consistency. Following vertical needle insertion, manual stimulation was applied using a standardized technique involving rapid bidirectional rotation (180° clockwise and counterclockwise) at a 2 Hz frequency for 1 minute. A calibrated temperature probe was heated to 47 $\pm$ 0.5 °C using a precision circulating water bath (set at 48 °C to maintain thermal stability). Following depilation, the probe was applied to the ST36 acupoint with consistent pressure for 1 minute, ensuring maintenance of the target temperature (46 °C) throughout the stimulation period. A capsaicin (CAP) solution was prepared in 99.5% ethanol (1% w/v). Sterile cotton applicators (0.2 mm diameter) were saturated with the solution and applied to the depilated ST36 acupoint for 1 minute, ensuring complete contact with the skin surface.

As illustrated in Fig. 1A, four different types of stimulation were applied sequentially to the ST36 acupoint of C57BL/6 mice: EA, MA, 46 °C, and CAP. The waveforms were subsequently compared before and during the stimulation.

Fig. 1.

Effects of diverse acupuncture-like stimuli at ST36 on colorectal motility in C57 mice. (A) Experimental flowchart for acupuncture-like stimulation at ST36 in C57 mice. Mouse image was modified from Heaster TM et al. (https://doi.org/10.3389/fbioe.2021.644648). (B–E) Representative example graphs of colorectal motility (left), and statistical graphs of AUC (middle) and amplitude (right) during EA (B), MA (C), 46 °C heat (D), and capsaicin (E) stimulation of C57 mice at ST36. Length of each red trace refers to 1 min. n = 6, *p 0.05, **p 0.01, ***p 0.001 vs. base. ST36, Zusanli acupoint; AUC, area under the curve; EA, electroacupuncture; MA, manual acupuncture.

Light (473/593 nm) for stimulation was delivered through a 200 µm diameter optical fiber (RWD, R-FC-L-N2-200-L1) positioned perpendicular to the skin and affixed tightly against it. The fiber tip was placed directly above the ST36 acupoint. Prior to each experiment, laser power output from the fiber optic cable was measured using a photometer (PM100D, Thor Labs, Newton, NJ, USA).

The optogenetic activation protocols shown in Fig. 3A employed identical motility recording and analysis pipelines as those established in prior experiments, with light parameters optimized to achieve suprathreshold TRPV1⁺ fiber activation (473 nm, 30 mW, 10 Hz).

For yellow light inhibition stimulation, after the colorectal motility recordings in mice had stabilized, a 1-minute recording segment was selected as the baseline for comparison. This was followed by a 1-minute EA (1 mA, 10 Hz) stimulation. Once the colorectal motility trace returned to a stable state, another 1-minute baseline was selected. Yellow light stimulation (20 mW, 10 Hz) was then applied for 30 seconds in advance, followed by a combined stimulation of yellow light (20 mW, 10 Hz) and EA (1 mA, 10 Hz) for 1 minute. The process was repeated with increasing intensities of yellow light, as shown in Fig. 6A. Changes in the AUC and amplitude of colorectal motility induced by EA alone and by the combination of yellow light and EA were compared. As shown in Fig. 7A, after determining the effective intensity of yellow light (30 mW, 10 Hz), different peripheral acupuncture-like stimuli were sequentially applied, with the same operational procedures and statistical parameters as described previously.

After recording of colorectal motility, TrpV1^ChR2-eYFP and TrpV1^NpHR-eYFP mice were weighed and anesthetized with 1.25% tribromoethanol (Avertin; 2 mL/100 g body weight, i.p.). TrpV1^𝐶𝑟𝑒 mice, which did not undergo motility recording, were weighed and anesthetized directly (Avertin; 2 mL/100 g body weight, i.p.). Following depilation of the ST36 acupoint region using electric clippers, thoracotomy was performed to expose the cardiac apex. Systemic perfusion was initiated with 40 mL of ice-cold phosphate-buffered saline (PBS; 0.9%, pH 7.4) followed by 40 mL of freshly prepared 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH 7.4), delivered at a rate of 5 mL/min using a peristaltic pump. Following perfusion, bilateral DRG from lumbosacral (L6–S2) segments were dissected and post-fixed in 4% PFA for 3 h at 4 °C. Tissues were then cryoprotected through sequential sucrose gradients (15% and 30% in 0.1 M PB) at 4 °C until equilibrium (typically 24–48 h), as determined by tissue sedimentation. Cryoprotected samples were stored in 30% sucrose solution at 4 °C until further processing.

Following complete cryoprotection, tissue samples were embedded in optimal cutting temperature (OCT) compound (Epredia Richard-Allan NEG 50, Kalamazoo, MI, USA) and rapidly frozen on a cryostat microtome freezing chamber (Leica CM1950, Wetzlar, Germany). Serial coronal sections (20 µm thickness) were obtained using a cryostat microtome maintained at –20 °C. Sections were systematically collected and mounted onto positively charged glass slides (Superfrost Plus, Thermo Fisher Scientific, Waltham, MA, USA). To ensure tissue integrity and minimize freeze-thaw artifacts, sections were air-dried for 30 minutes at room temperature.

Tissue sections were rehydrated in 0.1 M PB (pH 7.4) for 10 minutes to remove residual OCT compound. Non-specific binding sites were blocked with a solution containing 5% normal goat serum (Jackson ImmunoResearch, West Grove, CA, USA), 0.5% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA), and 0.1 M PB for 1 h at room temperature (22 $\pm$ 1 °C).

The following primary antibodies were diluted in antibody dilution buffer (3% normal goat serum, 0.5% Triton X-100 in 0.1 M PB) and applied to sections: TRPV1 (1:600, GP14100, NEUROM), CGRP (1:1500, 24112, IMMUNOSTAR), NF200 (1:600, Ab207176, Abcam), PER (1:1000, AVES, NEUROM), TH (1:600, ab112, Abcam), and GFP (1:1000, GFP-1010, Aves Labs). Incubation was performed in a humidified chamber at 4 °C for 16–18 h. After three 10-minute washes in 0.1 M PB, the following species-specific secondary antibodies conjugated with Alexa Fluor dyes (Thermo Fisher Scientific, Waltham, MA, USA) were applied at a 1:500 dilution in antibody dilution buffer and incubated for 2 h at room temperature in darkness: goat anti-guinea pig 594 (A-11076, ThermoFisher, Waltham, MA, USA), goat anti-chicken 488 (A-11039, ThermoFisher), and goat anti-rabbit 488 (A32731, ThermoFisher, Waltham, MA, USA). Following three washes, sections were then coverslipped using anti-fade mounting medium containing 50% glycerol (Vector Laboratories, San Francisco, CA, USA) and sealed with clear nail polish.

Fluorescence imaging was performed using an epifluorescence microscope (Olympus FV1200, Tokyo, Japan) equipped with appropriate filter sets and a confocal laser scanning microscope (Zeiss LSM 880, Oberkochen, Germany) with 20$\times$ and 40$\times$ objectives. Quantitative analysis of immunopositive cells was conducted using ImageJ software (v1.53, NIH, Bethesda, MD, USA).

All statistical analyses were performed using GraphPad Prism software (version 8.0, GraphPad Software Inc., San Diego, CA, USA). Data are expressed as the mean $\pm$ standard error of the mean (SEM), unless otherwise specified. Normality of data distribution was assessed using the Shapiro-Wilk test. For normally distributed paired data, parametric analysis was conducted using paired Student’s t-tests. Non-parametric data were analyzed using Wilcoxon signed-rank tests to evaluate the effects of stimulation on intestinal motility parameters. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for normally distributed data, or the Kruskal-Wallis test with Dunn’s correction for non-parametric data. The threshold for statistical significance was set at p 0.05. All statistical tests were two-tailed, with adjustment for multiple comparisons where appropriate.

We first systematically characterized the effects of diverse acupuncture-like stimuli modalities on colorectal motility patterns in C57BL/6 mice, focusing on the ST36 acupoint. The stimulation paradigm incorporated four distinct interventions, as illustrated in Fig. 1A.

Quantitative analysis of colorectal motility parameters (Fig. 1B–E) revealed that all stimulation modalities significantly enhanced motor function compared to baseline recordings. Specifically, marked increases were observed in the AUC (EA: from 214.8 $\pm$ 45.8 to 1022 $\pm$ 251.4, p = 0.0174; MA: from 246.5 $\pm$ 97.34 to 845.2 $\pm$ 220.8, p = 0.0313; 46 °C: from 147 $\pm$ 18.58 to 441.7 $\pm$ 90.22, p = 0.0313; CAP: from 111.4 $\pm$ 18.66 to 335.7 $\pm$ 131.9, p = 0.0313), as well as in contraction amplitude (EA: from 8.22 $\pm$ 2.19 to 33.83 $\pm$ 2.21, p = 0.0002; MA: from 9.15 $\pm$ 2.84 to 35.54 $\pm$ 4.54, p 0.0001; 46 °C: from 8.93 $\pm$ 1.65 to 24.58 $\pm$ 4.32, p = 0.0037; CAP: from 5.11 $\pm$ 0.77 to 16.96 $\pm$ 3.09, p = 0.0043). These findings indicate that multiple forms of acupuncture-like stimuli at ST36 can effectively modulate colorectal motility function in C57BL/6 mice.

In order to demonstrate the effectiveness of the TRPV1 channel in acupuncture-like stimuli, we conducted detailed immunohistochemical characterization of TRPV1-expressing neurons in DRG sections from TrpV1^𝐶𝑟𝑒 mice, focusing especially on the homosegemental level (L6–S2, Fig. 2). Quantitative analysis demonstrated that TRPV1 predominantly co-localized with markers of unmyelinated peptidergic neurons (CGRP: 27.39 $\pm$ 0.11%; PER: 46.76 $\pm$ 0.61%), while showing minimal overlap with NF200⁺ myelinated neurons (6.10 $\pm$ 3.23%) and TH⁺ sympathetic fibers (1%). This molecular profiling establishes TRPV1 as a marker of small-diameter, nociceptive neurons, prompting subsequent investigation of its functional involvement in acupuncture-mediated neural modulation.

Fig. 2.

Molecular characterization of TRPV1⁺ neurons in TrpV1^𝐶𝑟𝑒 mice DRG. (A–D) Representative immunofluorescence images showing the co-expression of TRPV1 (red) with: (A) CGRP (green), (B) NF200 (green), (C) Peripherin (green), and (D) Tyrosine hydroxylase (TH; green). Scale bar: left 100 µm, right 50 µm. Right panels: Quantification of co-localization percentages (mean $\pm$ SEM). Arrowheads indicate double stained neurons. TRPV1, transient receptor potential vanilloid 1; DRG, dorsal root ganglion; CGRP, calcitonin gene-related peptide; SEM, Standard error of the mean.

To determine whether TRPV1⁺ neurons are the best candidates for acupuncture effects, we generated TrpV1^ChR2-eYFP mice in which cutaneous TRPV1 sensory neurons can be selectively activated with precise temporal and spatial control using an external source of blue light. TrpV1^ChR2-eYFP mice were generated by breeding Ai32 mice [16] with mice expressing Cre recombinase under the control of the TRPV1 promoter [17]. ChR2-expressing neurons in DRG can be visualized using immunofluorescence microscopy based on their expression of eYFP. As expected, TRPV1⁺ neurons in the DRG of TrpV1^ChR2-eYFP mice co-expressed eYFP (Fig. 3A).

Fig. 3.

Effects of different intensities of blue light stimulation at ST36 on colorectal motility in TrpV1^ChR2-eYFP mice. (A) Immunofluorescent visualization of TrpV1^ChR2-eYFP DRG neurons, showing the expression of ChR2/eYFP (green) and TRPV1 (red) (top). Scale bar, 50 µm. Experimental flowchart for different intensities of blue light stimulation at ST36 in TrpV1^ChR2-eYFP mice (bottom). Arrowheads indicate double stained neurons. Mouse image was modified from Heaster TM et al. (https://doi.org/10.3389/fbioe.2021.644648). (B–D) Representative example graphs of colorectal motility (left), and statistical graphs of AUC (middle) and amplitude (right) during 20 mW (B), 30 mW (C), and 40 mW (D) blue light stimulation in TrpV1^ChR2-eYFP mice at ST36. Length of each red trace refers to 1 min. n = 5, *p 0.05, **p 0.01; ns, not significant, vs. base.

To determine the optimal optical stimulation parameters, we systematically evaluated the intensity of blue light (473 nm) on colorectal motility patterns in TrpV1^ChR2-eYFP transgenic mice using a graded intensity protocol ranging from subthreshold to suprathreshold levels (20–40 mW). As shown in Fig. 3B–D, optical stimulation was achieved at 473 nm with the parameters of 10 Hz frequency, 1 ms pulse width, and 1 min illumination duration. This elicited a significant increase in colorectal motility, as quantified by both AUC and contraction amplitude. Notably, light intensities of 30 mW and 40 mW produced robust and statistically significant responses (AUC: 20 mW, from 189.2 $\pm$ 38.44 to 161.6 $\pm$ 31.75, p = 0.0625; 30 mW, from 178.4 $\pm$ 60.43 to 306.2 $\pm$ 98.23, p = 0.0399; 40 mW, from 100.3 $\pm$ 13.62 to 215.4 $\pm$ 24.21, p = 0.0083. Contraction amplitude: 20 mW, from 6.28 $\pm$ 1.53 to 6.63 $\pm$ 1.46, p = 0.8125; 30 mW, from 8.15 $\pm$ 2.66 to 14.2 $\pm$ 3.13, p = 0.0059; 40 mW, from 4.58 $\pm$ 0.72 to 11.02 $\pm$ 1.17, p = 0.0185). Therefore, 30 mW was selected as the optimal stimulation intensity for subsequent optogenetic manipulations.

To examine the effects of the TrpV1 receptor during acupuncture-like stimulation, we next compared optogenetic stimulation (30 mW) with multiple acupuncture-like stimuli in TrpV1^ChR2-eYFP mice, as illustrated in Fig. 4A. Quantitative assessment of colorectal motility parameters (Fig. 4B–F) revealed that both optical stimulation and acupuncture-like interventions elicited significant potentiation of contractile activity, as evidenced by increased AUC and contraction amplitude. The remarkable similarity in response profiles provides compelling evidence that TRPV1-expressing sensory neurons mediate the visceromotor effects of acupuncture-like stimuli.

Fig. 4.

Colorectal motility of TrpV1^ChR2-eYFP mice in response to different acupuncture-like stimuli at ST36. (A) Experimental flowchart for different intensities of blue light stimulation at ST36 in TrpV1^ChR2-eYFP mice. Mouse image was modified from Heaster TM et al. (https://doi.org/10.3389/fbioe.2021.644648). (B–F) Representative example graphs of colorectal motility (left), and statistical graphs of AUC (middle) and amplitude (right) during stimulation of TrpV1^ChR2-eYFP mice at ST36 with 30 mW blue light (B), EA (C), MA (D), 46 ℃ heat (E), and CAP (F). Length of each red trace refers to 1 min. n = 8, *p 0.05, **p 0.01, ***p 0.001, ****p 0.001 vs. base.

Quantitative analysis also revealed distinct efficacy profiles among the stimulation modalities (Fig. 5). EA and MA interventions demonstrated superior modulation of colorectal motility function compared to 30 mW optogenetic stimulation (EA: 511.3 $\pm$ 125.9% vs. 30 mW blue light: 171.6 $\pm$ 57.84% of baseline AUC, p = 0.0423; EA: 454.9 $\pm$ 105.5% vs. 30 mW blue light: 179.3 $\pm$ 62.46% of baseline contraction amplitude, p = 0.0327; MA: 598.1 $\pm$ 136% vs. 30 mW blue light: 171.6 $\pm$ 57.84% of baseline AUC, p = 0.008; MA: 444.6 $\pm$ 93.95% vs. 30 mW blue light: 179.3 $\pm$ 62.46% of baseline contraction amplitude, p = 0.0415). In contrast, comparable efficacy profiles were observed for thermal stimulation at 46 °C (144.3 $\pm$ 30.71% of baseline AUC, p = 0.9985; 155.4 $\pm$ 18.98 of baseline contraction amplitude, p = 0.9976) and for capsaicin application (189 $\pm$ 55.34% of baseline AUC, p = 0.9998; 175.4 $\pm$ 32.43 of baseline contraction amplitude, p $>$ 0.9999), with no significant differences observed between these two modalities.

Fig. 5.

The extent of increased colorectal motility in TrpV1^ChR2-eYFP mice induced by different acupuncture-like stimuli at ST36. (A) Proportion of increase in the AUC of colorectal motility in TrpV1^ChR2-eYFP mice during stimulation at ST36 with blue light at 30 mW, EA, MA, 46 °C, and CAP, compared with the respective baselines. (B) Proportion of increase in the amplitude of colorectal motility in TrpV1^ChR2-eYFP mice during stimulation at ST36 with blue light at 30 mW, EA, MA, 46 °C, and CAP, compared with the respective baselines. n = 8, ^#p 0.05, ^#⁢#p 0.01; ns, not significant, vs. 30 mW.

We next employed an optogenetic loss-of-function strategy to investigate the contribution of TRPV1⁺ sensory pathways to acupuncture-evoked colorectal modulation. TrpV1^NpHR-eYFP enables cell-type-specific silencing of TRPV1⁺ afferents through yellow light (593 nm) activation of halorhodopsin. Immunohistochemical analysis of DRG sections (L6–S2) revealed precise co-localization of eNpHR3.0-eYFP expression with endogenous TRPV1 immunoreactivity, as shown in Fig. 6A.

Fig. 6.

Effects of EA and EA with different intensities of yellow light stimulation at ST36 on colorectal motility in TrpV1^NpHR-eYFP mice. (A) Immunofluorescent visualization of TrpV1^NpHR-eYFP DRG neurons showing the expression of NpHR/eYFP (green) and TRPV1 (red) (top). Scale bar, 50 µm. Experimental flowchart for different intensities of yellow light stimulation of ST36 in TrpV1^NpHR-eYFP mice (bottom). Arrowheads indicate double stained neurons. Mouse image was modified from Heaster TM et al. (https://doi.org/10.3389/fbioe.2021.644648). (B–D) Representative example graphs of colorectal motility (left) and statistical graphs of AUC (middle) and amplitude (right) during EA stimulation and EA with 20 mW (B), 30 mW (C) and 40 mW (D) of yellow light stimulation at ST36 in TrpV1^NpHR-eYFP mice. Length of each red trace refers to 1 min. n = 5, *p 0.05, **p 0.01 vs. EA; ns, not significant vs. EA.

To establish the optimal parameters for optogenetic inhibition, we systematically characterized the ideal intensity for yellow light (593 nm) stimulation of colorectal motility in TrpV1^NpHR-eYFP mice. As shown in Fig. 6B–D, quantitative analysis revealed that both 30 mW and 40 mW irradiance levels significantly suppressed colorectal motility function. This was evidenced by reduced AUC (30 mW: from 471 $\pm$ 91.24 to 105.8 $\pm$ 19.49 of baseline AUC, p = 0.0207; 40 mW: from 646.8 $\pm$ 153.8 to 109.7 $\pm$ 31.55, p = 0.0068) and contraction amplitude (30 mW: from 486.5 $\pm$ 103 to 160.9 $\pm$ 37.94, p = 0.0228; 40 mW: from 609.3 $\pm$ 95.84 to 205.3 $\pm$ 118.3, p = 0.0064). While the 20 mW irradiance level also reduced contraction amplitude, the change in colorectal motility AUC did not reach statistical significance (20 mW: from 351.6 $\pm$ 83.01 to 145.4 $\pm$ 28.65 of baseline AUC, p = 0.062; 20 mW: from 349.1 $\pm$ 47.91 to 183 $\pm$ 34.49 of baseline contraction amplitude, p = 0.0234). Based on comparable efficacy and reduced risk of tissue heating, 30 mW was selected as the standard inhibition parameter in subsequent experiments.

To further illustrate the inhibitory effect of TrpV1 during yellow light stimulation, we combined optogenetic inhibition with four distinct somatic acupuncture-like interventions in TrpV1^NpHR-eYFP mice (Fig. 7A). Detailed experimental procedures are described in the Methods section. As shown schematically in Fig. 7B–E and Fig. 8A,B, quantitative analysis demonstrated that optogenetic silencing of TRPV1⁺ afferents produced modality-specific attenuation of colorectal motility parameters. For AUC measurements, significant reductions in efficacy were observed across all stimulation conditions, as shown in Fig. 7 (EA: from 695.1 $\pm$ 136.4 to 274.3 $\pm$ 83.31, p = 0.0408; MA: from 518.4 $\pm$ 144.9 to 146 $\pm$ 15.32, p = 0.0479; 46 °C: from 347.8 $\pm$ 108.7 to 68.04 $\pm$ 15.3, p = 0.0464; CAP: from 683.6 $\pm$ 218.2 to 102.2 $\pm$ 32.57, p = 0.0434). Similarly, analysis of contraction amplitude revealed comparable inhibition profiles (EA: from 717.7 $\pm$ 113.6 to 279 $\pm$ 96.94, p = 0.0102; MA: from 585.4 $\pm$ 136 to 132.9 $\pm$ 51.05, p = 0.0407; 46 °C: from 400.9 $\pm$ 127.2 to 108.5 $\pm$ 31.66, p = 0.0431; CAP: from 856.5 $\pm$ 278.7 to 343.1 $\pm$ 248.8, p = 0.0196). These findings establish TRPV1⁺ sensory fibers as a critical neural substrate for somatic acupuncture stimulation-induced visceral modulation.

Fig. 7.

Effects of acupuncture-like stimuli and acupuncture-like stimuli combined with yellow light stimulation at ST36 on colorectal motility in TrpV1^NpHR-eYFP mice. (A) Experimental flowchart for four different stimuli and stimuli with 30 mW yellow light stimulation at ST36 in TrpV1^NpHR-eYFP mice. Mouse image was modified from Heaster TM et al. (https://doi.org/10.3389/fbioe.2021.644648). (B–E) Representative example graphs of colorectal motility (left), and statistical graphs of AUC (middle) and amplitude (right) of acupuncture-like stimuli: EA (B), MA (C), 46 ℃ heat (D), and CAP (E), and acupuncture-like stimuli with 30 mW yellow light stimulation in TrpV1^NpHR-eYFP mice at ST36. Length of each red trace refers to 1 min. n = 5, *p 0.05 vs. sti.

Fig. 8.

The proportion of contribution of TRPV1 to the facilitation of colorectal motility under various acupuncture-like stimuli at ST36. (A) Statistical chart of the proportion of TRPV1’s effect on the AUC of colorectal motility in TrpV1^NpHR-eYFP mice induced by EA, MA, 46 °C heat, and CAP stimulation at ST36. (B) Statistical chart of the proportional effect of TRPV1 on the amplitude of colorectal motility in TrpV1^NpHR-eYFP mice induced by EA, MA, 46 °C heat, and CAP stimulation at ST36. n = 5.

Further analysis revealed that TRPV1-expressing fibers at ST36 differentially mediate the effects of acupuncture-like stimuli modalities on colorectal motor parameters. EA: 64.97 $\pm$ 15.26% in AUC, 67.96 $\pm$ 13.55% in contraction amplitude; MA: 64.69 $\pm$ 7.11% in AUC, 71.09 $\pm$ 10.92% in contraction amplitude; 46 °C heat: 77.58 $\pm$ 3.89% in AUC, 64.49 $\pm$ 6.81% in contraction amplitude; CAP application: 81.95 $\pm$ 4.06% in AUC, 69.75 $\pm$ 11.43% in contraction amplitude.

Building on established neurophysiological principles in which A$\delta$/C-fiber co-activation enhances gastrointestinal motility [13, 18], we first verified that diverse somatosensory stimuli (EA, manual acupuncture, 46 °C heat, and capsaicin) at ST36 uniformly enhanced colorectal contractile activity in C57BL/6 mice.

TRPV1 serves as a polymodal transducer essential for peripheral nociception, and is therefore predominantly expressed in small- and middle-diameter peripheral neurons [19, 20]. Research has shown that its expression is enriched in acupoints and can be dynamically upregulated by EA [19]. In our model, TRPV1 showed preferential co-expression with CGRP⁺ nociceptors (20–25% overlap), but minimal co-localization with NF200⁺ neurons (6%), indicating predominant association with unmyelinated/thinly myelinated afferents [21]. Further immunohistochemical characterization demonstrates that peripherin (a peripheral neuron marker) colocalizes with TRPV1 in ~50% of small/medium neurons, confirming predominant peripheral nociceptor expression. Tyrosine hydroxylase (TH), a marker for C-fiber low-threshold mechanoreceptors (on average, 15% of mouse DRG neurons express TH) [22], shows no co-expression with TRPV1. Collectively, these findings establish TRPV1 as a selective biomarker for small-to-medium diameter nociceptive neurons in somatosensory pathways.

Specific optogenetic activation of TRPV1⁺ afferents with blue light (473 nm) enhanced colorectal motility in TrpV1^ChR2-eYFP mice. The effect was comparable in amplitude to that induced by 46 °C heat or capsaicin interventions, but was less potent than EA or MA (Fig. 5). This mechanistic divergence arises from differential nociceptor engagement profiles, with 46 °C heat, capsaicin and optogenetic stimulation primarily activating cutaneous TRPV1⁺ fibers, whereas EA and MA concurrently engage multiple nociceptor subtypes (e.g., TRPV1⁺, ASIC3⁺, P2X3⁺ populations) across cutaneous and muscular compartments, generating synergistic neuromodulatory effects through spatial summation in spinal cord projection neurons [23].

Conversely, optogenetic inhibition of TRPV1⁺ afferents during EA at ST36 with yellow light (589 nm) in TrpV1^NpHR-eYFP mice significantly attenuated the stimulation-induced potentiation of colorectal motility by various modalities (Figs. 6,7). Consistent with previous findings in TRPV1 KO mice, our results support the crucial role of TRPV1 in mediating acupuncture-like stimuli effects [24]. Notably, Dong et al. [25] recently provided direct functional evidence by demonstrating that specific pharmacological inhibition of deep TRPV1⁺ sensory fibers innervating the ST36 region prevented EA at ST36 from effectively triggering gastric reflexes. This finding suggests that deep TRPV1⁺ sensory fibers innervating the ST36 fascial compartment are one of the key initial components in the cascade of EA-promoted gastric motility [25]. As shown in Fig. 8, we observed variations in the relative contribution of TRPV1-mediated signaling to colorectal motility regulation depending on the stimulus. This indicates that TRPV1 plays a complex role in mediating the regulatory effects of acupuncture-like stimuli on colorectal motility, with variable contributions depending on the type of stimulation and the specific metrics measured. While optogenetic silencing of TRPV1⁺ fibers at the ST36 acupoint was utilized in this study, future validation should incorporate orthogonal approaches. This could include topical application of selective antagonists, and chemogenetic inhibition via DREADD-expressing models to establish modality-independent causality.

A key consideration is how activation of TRPV1⁺ afferents in the hindlimb (ST36) influences distant visceral motility. The somato-visceral pathway recruited by ST36 stimulation likely involves both spinal and vagal mechanisms. Afferents from the ST36 region (tibialis anterior muscle) primarily originate from lumbar DRGs (L4–L6) and project to the corresponding spinal segments [26]. Following initial integration in the spinal cord, this information may influence colorectal motility via a spino-bulbo-vagal pathway [24], reaching brainstem nuclei such as the nucleus tractus solitarius and the dorsal motor nucleus of the vagus, ultimately modulating gastrointestinal function through vagal efferents. Supporting vagal involvement, optogenetic stimulation of ST36 has been shown to robustly potentiate cervical vagal efferent activity. Thus, the observed modulation of colorectal motility may result from activation of TRPV1⁺ lumbar afferents at ST36 and their subsequent integration within this spino-vagal axis.

While optogenetics provides cell-type-specific causality, several limitations should be considered. (1) The penetration depth of transdermal optical stimulation is limited, likely affecting cutaneous and superficial subcutaneous fibers [27, 28]. (2) Experiments were conducted under isoflurane anesthesia, which may influence autonomic tone and baseline colonic motility [29]. (3) While our motility data support effective inhibition by yellow light, we lack in vivo electrophysiological recordings (e.g., from the sciatic nerve or dorsal roots) to directly quantify the silencing efficiency of TRPV1⁺ fibers during NpHR activation. (4) Sample sizes were small for some experimental groups. Future studies should employ orthogonal approaches—such as topical application of selective antagonists and chemogenetic inhibition—for validation, and directly investigate the complete neural circuitry in awake, behaving models.