Specific pathogen-free (SPF) grade C57BL/6J male mice (4–8 weeks old) were purchased from the Shanghai Tongji Biotechnology Co., Ltd. (Shanghai, China). All mice were kept in a room with controlled temperature (25 ± 1°C) and humidity (55 ± 5%), and 12 h (8:00 a.m. to 8:00 p.m.) light–dark cycle with free access to water and rodent chow. All mice were group-housed (4–6 per cage) to avoid social isolation stress. All experimental protocols were approved by the Ethics Committee of the Laboratory Animal Center of Tongji University (Approval Number: TJBH05621101) and performed following the Guide for the Care and Use of Laboratory Animals of Tongji University (Shanghai, China) and the International Guide for the Care and Use of Laboratory Animals.

Retrograde tracer in this study included Fluoro-Gold (FG, Fluorochrome Inc., Englewood, CO, United States) and the lipophilic tracer, 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, D282; Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) (Chang et al., 2016). FG was dissolved in 0.9% saline to yield a 4% concentration (Szawka et al., 2013; Pattwell et al., 2016). DiI was dissolved in dimethyl sulfoxide (DMSO, D2650; Sigma-Aldrich, St. Louis, MO, United States) as a stock solution, and freshly prepared in 0.9% saline.

During DiI stereotaxic injections, the mice were fixed in mouse pockets and held in the appropriate position of the stereotaxic apparatus. The left leg that needed to be injected was secured to the outer wall of the mouse pocket. The injection site was ST36 acupoint, so fur around ST36 acupoint needs to be shaved to expose the skin. ST36 acupoint is 2–3 mm below the small head of the fibula and lateral to the anterior tubercle of the tibia (Zhang K. et al., 2018; Lin et al., 2019). According to the location of the ST36 acupoint, the tip of the injection needle was first positioned on the skin surface of the small head of the fibula as the starting points (lateral 0 mm, longitudinal 0 mm, and depth 0 mm). Then, 5 μL of DiI (2 mg/mL) was injected unilaterally into ST36 acupoint (lateral 2.5 mm, longitudinal 0 mm, and depth 3 mm) at a flow rate of 1 μL/min by a monitored microsyringe. The needle stayed at ST36 acupoint for an extra 1 min before being withdrawn slowly. Ten days after DiI injection, 1.5 μL FG was injected into the plantar surface of the left hind paw of mice. Two weeks after DiI injection and 4 days after FG injection, lumbar 4 to lumbar 6 (L4–L6) DRGs were dissected for immunofluorescence. A total of 15 mice were used in this experiment.

Viruses utilized in this study included AAVRetro-hSyn-DIO-hM4D(Gi)-mCherry (titer 1.060 × 10¹² virus particles/mL), AAVRetro-hSyn-DIO-hM3D(Gq)-mCherry (titer 1.135 × 10¹² virus particles/mL), AAVRetro-hSyn-DIO-mCherry (titer 1.163 × 10¹² virus particles/mL), and AAVRetro-hSyn-Cre-EGFP (titer 1.285 × 10¹² virus particles/mL), all of which were obtained from the Shanghai Genechem Technology Co., Ltd. Viruses were injected 3–4 weeks before behavioral tests.

ST36 acupoint stereotaxic injection was performed as previously described. Eight microliters of AAVRetro-hSyn-DIO-hM4D(Gi)-mCherry, AAVRetro-hSyn-DIO-hM3D(Gq)-mCherry or AAVRetro-hSyn-DIO-mCherry was injected into left ST36 acupoint (lateral 2.5 mm, longitudinal 0 mm, sand depth 3 mm) at a flow rate of 1.5 μL/min. And 8 μL of AAVRetro-hSyn-Cre-EGFP was injected into the plantar surface of the left hind paw of mice who were fixed in mouse pockets during the injection. During all injections, the needle was allowed to remain at the target site for an additional 10 min and then removed slowly to prevent efflux of the virus. All virus injections were completed on the same day. A total of 85 mice received virus injections.

For hM4Di silencing or hM3Dq activation, mice were injected clozapine-N-oxide (CNO, C0832, Sigma-Aldrich) intraperitoneal (i.p.) at 5 mg/kg, 30 min before the behavioral test (Stoodley et al., 2017; Liu et al., 2020). CNO was dissolved in DMSO at 10 mg/mL as a stock solution, kept at – 20°C until used, and freshly diluted with 0.9% saline to the corresponding working concentrations in a final volume of 300 μL before intraperitoneal administration. The final concentration of DMSO in working solutions did not exceed 0.1% (vol/vol). Saline was injected as vehicle control.

Fifty mice received CFA injections. CFA was purchased from Sigma-Aldrich (F5881), which contains 1 mg/mL of heat-killed and dried mycobacterium tuberculosis in 85% paraffin oil and 15% mannide monooleate. Unilateral inflammation pain was induced by injecting 20 μL CFA into the plantar surface of the left hind paw of mice who were fixed in mouse pockets during the injection (Wu et al., 2019). Once finished injection, the injection site was immediately pressed gently with cotton to prevent bleeding and drug leakage.

Mechanical pain threshold (MPT) was assessed with the electronic Von Frey test (BIO-EVF4, Bioseb, Vitrolles, France) at three locations separately, ST36 acupoint, 5 mm lateral side of ST36 acupoint (LST), and plantar of the IHP. To measure the MPT of IHP, each mouse was placed in a suspended plastic cage (66 × 22 × 13 cm) with a wire grid floor (0.6 × 0.6 cm² spacing). And to measure the MPT of ST36 acupoint or LST, the mice were fixed in mouse pockets. The electronic von Frey apparatus, consisting of an elastic spring-type tip fitted in a hand-held force transducer, was applied perpendicularly to the skin surface of the three locations and the force applied was gradually increased until hind paw withdrawal. Finally, MPT (expressed in grams) was automatically recorded by the electronic Von Frey device and regarded as a pain parameter (Ferrier et al., 2013).

Thermal pain threshold (TPT) was evaluated with a thermal pain test apparatus (Hargreaves Apparatus, No. 37370, Ugo Basile, Comerio, Italy). Before testing, mice were individually kept in a transparent plastic test chamber (62 × 20 × 14 cm) fixed in the proper position on a suspended, specially constructed, and non-insulated transparent glass panel (86 × 35 cm). The intensity of the infrared radiant heat source (IR value) was adjusted and kept constant. And to prevent possible tissue damage, the basal TPT was adjusted to 10–12 s and a cutoff of 20 s (Mao-Ying et al., 2012; Feng et al., 2019). In this study, a movable stimulus of infrared radiant heat (IR = 23) was placed under the glass floor directly beneath the middle plantar surface of the left hind paw. When the withdrawal response occurred, the IR source switched off, and the reaction time counter stopped. The TPT was automatically detected and recorded (in seconds) with an accuracy of 0.1 s.

DRGs (L4-L6) were harvested from 15 mice perfused with ice-cold PBS and 4% paraformaldehyde (PFA). DRGs were fixed at 4°C in 4% PFA for at least 2 h, incubated in 30% sucrose overnight at 4°C, and embedded in optimal cutting temperature compound (OCT, 4583, Tissue-Tek, SAKURA, Torrance, CA, United States). Coronal sections (30 μm thick) were cut with a cryomicrotome (CM 1950, Leica Microsystems, Nussloch, Germany), and adhered to slides. For staining, sections were washed in PBS three times (3 min for each wash), blocked in PBS containing 5% normal goat serum for 2 h at room temperature, and incubated with primary antibodies overnight at 4°C. Primary antibodies utilized in this study included rabbit polyclonal anti-transient receptor potential vanilloid 1 (TRPV1) antibody (1:400 dilution, ab6166, Abcam, Cambridge, United Kingdom), and rabbit monoclonal anti-Parvalbumin (PV) Antibody (1:200 dilution, MA5-35259, Thermo Fisher Scientific). After three washes with PBS for 3 min each time, the sections were incubated with goat polyclonal secondary antibody to rabbit IgG-H&L (Alexa Fluor^® 488, 1:500 dilution, ab150077, Abcam) for 2 h at room temperature. Finally, sections were washed in PBS three times (3 min for each wash) and sealed with an appropriate amount of antifading mounting reagent (S2100, Solarbio, Shanghai, China). The images were captured by confocal laser-scanning microscopy using an Olympus FluoView FV3000 self-contained confocal laser scanning microscope system (Olympus, Tokyo, Japan). The data of immunofluorescent staining presented in the Figures 2A, 3A, 7A,D are representative images of at least five mice (Rezende et al., 2017; Lavian and Korngreen, 2019).

The statistical analysis was performed using the Graphpad Prism 8 software (GraphPad, San Diego, CA, United States). All data are presented as mean ± standard deviation (SD) and checked for normality before analysis. For comparison of parameters between two groups (Figures 1, 3B,C, 4–6), the variance similarity was determined by the F-test. And significance was determined by paired t-test. For all tests, differences of P < 0.05 were considered to be statistically significant. Statistical analysis of individual experiments is also described in figure legends.

The acupoint being more sensitive to external mechanical stimuli is one of the characteristics of acupoint sensitization. After the CFA model was confirmed to be built successfully (Supplementary Figure 1), we checked the pain threshold of hind paw plantar in the CFA-treated mice. After 4 days of CFA injection into the hind paw plantar, the MPTs of the hind paws were decreased from 4.8 ± 0.3 g to 1.9 ± 0.3 g (Figure 1A), and the TPTs were reduced from 12.7 ± 0.4 s to 2.8 ± 0.9 s (Figure 1B). These indicate that we achieved a stable CFA pain model. Then we evaluated the MPTs of ipsilateral ST36 acupoints in these CFA model mice by the von Frey method. CFA treatment significantly reduced the MPTs from 8.4 ± 0.4 g to 5.6 ± 1.1 g (Figure 1C), while the thresholds of the position 5 mm lateral side of the ST36 (LST) did not change much, from 8.3 ± 0.4 g to 8.4 ± 0.5 g (Figure 1D). These data suggested that ST36 acupoint was sensitized in the CFA-induced inflammatory pain state.

Since the sensory afferents of the calf and foot are the component of the sciatic nerve, and our previous study also showed that the somata of the nerve terminals innervating the ST36 acupoint were distributed in the L4–L6 DRG (Li et al., 2022), we hypothesized that there may be DRG neurons innervating both the plantar and ipsilateral ST36 acupoint in naïve mice. We retro-labeled DRG neurons innervating ST36 acupoint and ipsilateral plantar by injecting DiI or FG dyes at these two sites, respectively (Figure 2A). Since we are more concerned about how many neurons related to ST36 acupoint belong to shared neurons, we used the number of DiI-positive neurons as the denominator and the number of double-labeled neurons as the numerator when calculating the percentage, that is (DiI⁺ FG⁺)/DiI⁺ × 100%. After statistical analysis, we found that about 47.02% (1,056/2,246) of DRG neurons were double-labeled by DiI and FG, which indicated that these neurons were shared by ST36 acupoint and IHP plantar (Figure 2B). Of these shared neurons, 43.66% (461/1,056) were small-diameter neurons, 40.72% (430/1,056) were middle-size neurons, and only 15.63% (165/1,056) were large-diameter neurons (Figure 2C).

To understand the potential function of shared DRG neurons, we injected the AAVRetro-hSyn-Cre-EGFP virus into hind paw plantar and AAVRetro-hSyn-DIO-hM4D(Gi)-mCherry virus into ipsilateral ST36 acupoint at the same time to induce the expression of Gi-coupled human M4 muscarinic DREADD receptors in the shared DRG neurons. By immunofluorescence method, mCherry signals were detected in many DRG neurons (Figure 3A), which indicated that two viruses infected DRG terminals at two separate positions were retrogradely transported to the same soma, and expressed mCherry in a cre-dependent manner. Then CNO was injected intraperitoneally to specifically inhibit shared DRG neurons. We compared the MPTs and the TPTs after CNO treatment with those before CNO treatment and found that, in the CFA model, both thresholds were elevated significantly (MPTs from 1.9 ± 0.2 g to 5.0 ± 0.4 g, TPTs from 3.1 ± 1.1 s to 8.5 ± 2.7 s) (Figure 3B). Even in normal mice, the thresholds were also increased a little (MPTs from 4.9 ± 0.3 g to 5.5 ± 0.6 g, TPTs from 12.6 ± 0.6 s to 14.5 ± 1.4 s) (Figure 3C). These data suggest that selectively inhibition of shared DRG neurons induced analgesic effect in the CFA pain model and obstruction of nociceptive sensation in normal mice. We also performed the same studies as shown in Figure 3, except that the AAVRetro-hSyn-DIO-hM4D(Gi)-mCherry virus was replaced by the AAVRetro-hSyn-DIO-mCherry virus, which means that only the mCherry proteins, but not M4 muscarinic DREADD receptors, were expressed in shared DRG neurons. The data showed that CNO did not change the pain thresholds of plantar both in the CFA model and in normal mice (Supplementary Figures 2A,B), which indicates that the change of pain thresholds was dependent on the expression of M4 muscarinic DREADD receptors in the shared DRG neurons, in other words, the inhibition of shared DRG neurons.

As the shared DRG neurons innervate both hind paw plantar and ST36 acupoint, they may be involved in the sensation of both locations. We hypothesize that inhibition of these neurons might also block the pain sensation of the ipsilateral ST36 acupoint as they did of the hind paw. In the CFA model, the MPTs of ST36 elevated from 5.0 ± 1.1 g to 7.7 ± 0.1 g by CNO treatment, while those of the LST position did not change much, from 8.4 ± 0.5 g to 8.6 ± 0.2 g (Figure 4A). In normal mice, the thresholds of LST also did not change by CNO, from 8.3 ± 0.4 g to 8.3 ± 0.1 g, but those of the ST36 still increased a little, from 8.4 ± 0.4 g to 9.4 ± 0.6 g (Figure 4B). The changing trend of MPTs at the ST36 acupoint was quite like those at the ipsilateral plantar.

The results of inhibition of shared DRG neurons indicate that these neurons are necessary conditions for, or at least involved in, transmitting nociceptive pain information from paw plantar or ST36 acupoint to the spinal cord. Then, we performed similar chemogenetic experiments to activate the shared DRG neurons specifically and evaluated the pain thresholds of the hind paw. The AAVRetro-hSyn-Cre-EGFP virus and AAVRetro-hSyn-DIO-hM3D(Gq)-mCherry virus were injected into hind paw plantar and ipsilateral ST36 acupoint separately at the same time. The CNO was injected intraperitoneally to specifically excite shared DRG neurons. The MPTs and the TPTs after CNO treatment were compared with those before CNO treatment. In normal mice, both thresholds declined significantly (MPTs from 5.1 ± 0.3 g to 2.6 ± 0.6 g, TPTs from 12.6 ± 0.5 s to 6.8 ± 2.1 s) (Figure 5A). These data suggest that selective activation of shared DRG neurons was sufficient to induce pain. But in the CFA model, the thresholds were not changed much (MPTs from 1.9 ± 0.3 g to 1.9 ± 0.4 g, TPTs from 2.6 ± 0.8 s to 2.6 ± 0.7 s) (Figure 5B).

Furthermore, we examined the change in the MPT of ST36 acupoint when shared DRG neurons were activated by CNO. In normal mice, the MPTs of ST36 declined from 8.2 ± 0.2 g to 6.6 ± 0.3 g by CNO treatment, while those of the LST position did not change much, from 8.3 ± 0.2 g to 8.4 ± 0.2 g (Figure 6A). In the CFA model, the thresholds of LST also did not change by CNO, from 8.6 ± 0.4 g to 8.6 ± 0.1 g, while those of the ST36 still decreased a little, from 6.1 ± 0.7 g to 5.4 ± 0.5 g (Figure 6B). The changing trend of the MPTs at ST36 acupoint was also quite like those at the ipsilateral plantar.

We also performed the same studies as in Figure 6, except that the AAVRetro-hSyn-DIO-hM3D(Gq)-mCherry virus was replaced by the AAVRetro-hSyn-DIO-mCherry virus, which means that only the mCherry proteins, but not M3 muscarinic DREADD receptors, were expressed in shared DRG neurons. The data showed that CNO did not change the pain thresholds of ST36 acupoint or LST both in normal mice and in the CFA model (Supplementary Figures 3A,B), which indicates that the change of pain thresholds of ST36 acupoint in normal mice was dependent on the expression of M3 muscarinic DREADD receptors in the shared DRG neurons, in other words, the activation of shared DRG neurons.

Based on the cell-size ratio of shared DRG neurons, most neurons were small to middle size. And the results of chemogenetic experiments strongly suggest that they are necessary and sufficient for the transmission of pain signaling. Thus, we stained the DRG slices with TRPV1 or PV antibodies to verify the expression properties of these neurons. About 62.93% (382/607) of shared DRG neurons were TRPV1⁺ neurons (Figures 7A,B), of which about 83.51% neurons were small (149/382) or middle (170/382) size neurons (Figure 7C). About 36.97% (166/449) of shared DRG neurons were PV⁺ neurons (Figures 7D,E). The 69.88% of PV⁺ neurons were large (51/166) or middle (65/166) size neurons (Figure 7F). These results went along that most of the shared DRG neurons were nociceptive neurons.