Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A neuroanatomical basis for electroacupuncture to drive the vagal–adrenal axis

Abstract

Somatosensory autonomic reflexes allow electroacupuncture stimulation (ES) to modulate body physiology at distant sites1,2,3,4,5,6 (for example, suppressing severe systemic inflammation6,7,8,9). Since the 1970s, an emerging organizational rule about these reflexes has been the presence of body-region specificity1,2,3,4,5,6. For example, ES at the hindlimb ST36 acupoint but not the abdominal ST25 acupoint can drive the vagal–adrenal anti-inflammatory axis in mice10,11. The neuroanatomical basis of this somatotopic organization is, however, unknown. Here we show that PROKR2Cre-marked sensory neurons, which innervate the deep hindlimb fascia (for example, the periosteum) but not abdominal fascia (for example, the peritoneum), are crucial for driving the vagal–adrenal axis. Low-intensity ES at the ST36 site in mice with ablated PROKR2Cre-marked sensory neurons failed to activate hindbrain vagal efferent neurons or to drive catecholamine release from adrenal glands. As a result, ES no longer suppressed systemic inflammation induced by bacterial endotoxins. By contrast, spinal sympathetic reflexes evoked by high-intensity ES at both ST25 and ST36 sites were unaffected. We also show that optogenetic stimulation of PROKR2Cre-marked nerve terminals through the ST36 site is sufficient to drive the vagal–adrenal axis but not sympathetic reflexes. Furthermore, the distribution patterns of PROKR2Cre nerve fibres can retrospectively predict body regions at which low-intensity ES will or will not effectively produce anti-inflammatory effects. Our studies provide a neuroanatomical basis for the selectivity and specificity of acupoints in driving specific autonomic pathways.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Characterizing PROKR2ADV neurons.
Fig. 2: Requirement of PROKR2ADV neurons for low-intensity ES to drive the vagal–adrenal anti-inflammatory axis.
Fig. 3: Activation of PROKR2ADV fibres drives the vagal–adrenal anti-inflammatory axis.
Fig. 4: Requirement of deep-tissue-innervating nerves for anti-inflammatory effects.

Data availability

All data are included in the paper and available from the corresponding author upon request. Source data are provided with this paper.

References

  1. 1.

    Kametani, H., Sato, A., Sato, Y. & Simpson, A. Neural mechanisms of reflex facilitation and inhibition of gastric motility to stimulation of various skin areas in rats. J. Physiol. 294, 407–418 (1979).

    CAS  Article  Google Scholar 

  2. 2.

    Sato, A. & Schmidt, R. F. The modulation of visceral functions by somatic afferent activity. Jpn. J. Physiol. 37, 1–17 (1987).

    CAS  Article  Google Scholar 

  3. 3.

    Sato, A. Neural mechanisms of autonomic responses elicited by somatic sensory stimulation. Neurosci. Behav. Physiol. 27, 610–621 (1997).

    CAS  Article  Google Scholar 

  4. 4.

    Li, Y. Q., Zhu, B., Rong, P. J., Ben, H. & Li, Y. H. Neural mechanism of acupuncture-modulated gastric motility. World J. Gastroenterol. 13, 709–716 (2007).

    Article  Google Scholar 

  5. 5.

    Takahashi, T. Effect and mechanism of acupuncture on gastrointestinal diseases. Int. Rev. Neurobiol. 111, 273–294 (2013).

    Article  Google Scholar 

  6. 6.

    Ma, Q. Somato–autonomic reflexes of acupuncture. Med. Acupunct. 32, 362–366 (2020).

    Article  Google Scholar 

  7. 7.

    Chavan, S. S., Pavlov, V. A. & Tracey, K. J. Mechanisms and therapeutic relevance of neuro-immune communication. Immunity 46, 927–942 (2017).

    CAS  Article  Google Scholar 

  8. 8.

    Ulloa, L., Quiroz-Gonzalez, S. & Torres-Rosas, R. Nerve stimulation: immunomodulation and control of inflammation. Trends Mol. Med. 23, 1103–1120 (2017).

    Article  Google Scholar 

  9. 9.

    Pan, W. X., Fan, A. Y., Chen, S. & Alemi, S. F. Acupuncture modulates immunity in sepsis: toward a science-based protocol. Auton. Neurosci. 232, 102793 (2021).

    CAS  Article  Google Scholar 

  10. 10.

    Torres-Rosas, R. et al. Dopamine mediates vagal modulation of the immune system by electroacupuncture. Nat. Med. 20, 291–295 (2014).

    CAS  Article  Google Scholar 

  11. 11.

    Liu, S. et al. Somatotopic organization and intensity dependence in driving distinct NPY-expressing sympathetic pathways by electroacupuncture. Neuron 108, 436−435 (2020).

    CAS  Article  Google Scholar 

  12. 12.

    Longhurst, J. C. Defining meridians: a modern basis of understanding. J. Acupunct. Meridian Stud. 3, 67–74 (2010).

    Article  Google Scholar 

  13. 13.

    Yang, F. C. et al. Genetic control of the segregation of pain-related sensory neurons innervating the cutaneous versus deep tissues. Cell Rep. 5, 1353–1364 (2013).

    CAS  Article  Google Scholar 

  14. 14.

    Choi, S. et al. Parallel ascending spinal pathways for affective touch and pain. Nature 587, 258–263 (2020).

    ADS  CAS  Article  Google Scholar 

  15. 15.

    Zylka, M. J., Rice, F. L. & Anderson, D. J. Topographically distinct epidermal nociceptive circuits revealed by axonal tracers targeted to Mrgprd. Neuron 45, 17–25 (2005).

    CAS  Article  Google Scholar 

  16. 16.

    Ghitani, N. et al. Specialized mechanosensory nociceptors mediating rapid responses to hair pull. Neuron 95, 944–954.e4 (2017).

    CAS  Article  Google Scholar 

  17. 17.

    Sharma, N. et al. The emergence of transcriptional identity in somatosensory neurons. Nature 577, 392–398 (2020).

    ADS  CAS  Article  Google Scholar 

  18. 18.

    Kupari, J. et al. Single cell transcriptomics of primate sensory neurons identifies cell types associated with chronic pain. Nat. Commun. 12, 1510 (2021).

    ADS  CAS  Article  Google Scholar 

  19. 19.

    Kucera, J. & Walro, J. M. An immunocytochemical marker for early type I muscle fibers in the developing rat hindlimb. Anat. Embryol. (Berl.) 192, 137–147 (1995).

    CAS  Google Scholar 

  20. 20.

    Remick, D. G., Newcomb, D. E., Bolgos, G. L. & Call, D. R. Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture. Shock 13, 110–116 (2000).

    CAS  Article  Google Scholar 

  21. 21.

    Kleinlogel, S. et al. Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh. Nat. Neurosci. 14, 513–518 (2011).

    CAS  Article  Google Scholar 

  22. 22.

    Lima, D. in The Senses: A Comprehensive Reference Vol. 5 (eds Masland, R. H. et al.) 477–526 (Academic, 2008).

  23. 23.

    Travagli, R. A. & Anselmi, L. Vagal neurocircuitry and its influence on gastric motility. Nat. Rev. Gastroenterol. Hepatol. 13, 389–401 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Damarey, B. et al. Imaging of the nerves of the knee region. Eur. J. Radiol. 82, 27–37 (2013).

    CAS  Article  Google Scholar 

  25. 25.

    Quiroz-Gonzalez, S., Segura-Alegria, B., Guadarrama-Olmos, J. C. & Jimenez-Estrada, I. Cord dorsum potentials evoked by electroacupuncture applied to the hind limbs of rats. J. Acupunct. Meridian Stud. 7, 25–32 (2014).

    Article  Google Scholar 

  26. 26.

    Peng, Z., Nan, G., Cheng, M. & Zhou, K. The comparison of trigger point acupuncture and traditional acupuncture. World J. Acupunct. Moxibustion 26, 1–6 (2016).

    Article  Google Scholar 

  27. 27.

    Choi, E. M., Jiang, F. & Longhurst, J. C. Point specificity in acupuncture. Chin. Med. 7, 4 (2012).

    Article  Google Scholar 

  28. 28.

    Xing, J. J., Zeng, B. Y., Li, J., Zhuang, Y. & Liang, F. R. Acupuncture point specificity. Int. Rev. Neurobiol. 111, 49–65 (2013).

    Article  Google Scholar 

  29. 29.

    Langevin, H. M. & Wayne, P. M. What is the point? The problem with acupuncture research that no one wants to talk about. J. Altern. Complement. Med. 24, 200–207 (2018).

    Article  Google Scholar 

  30. 30.

    van der Poll, T., van de Veerdonk, F. L., Scicluna, B. P. & Netea, M. G. The immunopathology of sepsis and potential therapeutic targets. Nat. Rev. Immunol. 17, 407–420 (2017).

    Article  Google Scholar 

  31. 31.

    Gerfen, C. R., Paletzki, R. & Heintz, N. GENSAT BAC Cre-recombinase driver lines to study the functional organization of cerebral cortical and basal ganglia circuits. Neuron 80, 1368–1383 (2013).

    CAS  Article  Google Scholar 

  32. 32.

    Bourane, S. et al. Identification of a spinal circuit for light touch and fine motor control. Cell 160, 503–515 (2015).

    CAS  Article  Google Scholar 

  33. 33.

    Liu, Y. et al. VGLUT2-dependent glutamate release from peripheral nociceptors is required to sense pain and suppress itch. Neuron 68, 543–556 (2010).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank D. D. Ginty for critical comments, and W. Lu and B. Zhao for helpful discussions; D. D. Ginty, M. Goulding, S. M. Dymecki, staff at GENSAT/MMRRC at the University of Davis, and the Allen Brain Institute/the Jackson Laboratory for genetically modified mice; and S. Celine for her assistance in histochemical analyses. All experimental data were generated at the Dana-Farber Cancer Institute, and the work was supported primarily by a NIH grant (R01AT010629) and partially by the Harvard/MIT Joint Research Program in Basic Neuroscience and a Wellcome Trust grant (200183/Z/15/Z) to Q.M. S.L.’s salary was supported primarily by a NIH grant (R01AT010629) and partly by the China Postdoctoral Science Foundation (KLF101846) and by the Development Project of Shanghai Peak Disciplines-Integrated Medicine (20150407) during an early period before the NIH grant (R01AT010629) was funded. Y.S.’s and W.Y.’s salary was supported by the China Scholarship Council (CSC no. 201609110039 and CSC no. 20190600178, respectively). Z.W.’s salary was supported by Fujian University of Traditional Chinese Medicine and by the Harvard/MIT Joint Research Program in Basic Neuroscience. Histochemical imaging was supported by Boston Children’s Hospital IDDRC (1U54HD090255).

Author information

Affiliations

Authors

Contributions

S.L. and Z.W. jointly performed most experiments. Y.S. helped to determine electric stimulation intensities for driving distinct autonomic pathways. L.Q. helped to characterize the identities of PROKR2Cre-marked sensory neurons. W.Y. determined differential representations of PROKR2Cre-marked DRG neurons at differential axial levels, and M.F. helped to show innervation of PROKR2Cre-marked sensory neurons in fascial tissues. Q.M. conceptualized and supervised the entire study (with contributions from Y.W. and X.J.). S.L. and Q.M. wrote the manuscript, and all authors edited the manuscript.

Corresponding author

Correspondence to Qiufu Ma.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Patrik Ernfors, Kevin Tracey, Luis Ulloa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Intersectional marking and characterization of PROKR2ADV neurons.

a, Schematic description of the intersectional genetic strategy for generating Prokr2Adv-tdTomato mice, in which tdTomato expression was confined to PROKR2ADV sensory neurons upon removal of two STOP cassettes from the ROSA26 allele by the Cre and Flpo recombinases. AdvillinFlpo drove Flpo expression restricted to sensory neurons, such that DRG neurons with developmental coexpression of PROKR2-Cre and ADVILLIN-Flpo were marked by tdTomato. b, Representative sections through suprarenal sympathetic ganglia, adrenal medulla where chromaffin cells were located, the colon that should contain enteric ganglia, and nodose ganglia from Prokr2Adv-tdTomato mice. The intersectional genetic strategy only labeled one or two cells per section in nodose ganglia. Arrows indicate tdTomato+ fibers, but not somas, likely from DRG neurons or from those rare nodose sensory neurons. In other words, PROKR2ADV-tdTomato did not label any autonomic neurons and adrenal chromaffin cells. n = 5 mice per group. c, L4-5 DRG sections from adult Prokr2Adv-tdTomato mice. Top panels show double immunostaining of tdTomato (red) with markers for nonperptidergic neurons (IB4: isolectin B4), proprioceptors (PV: parvalbumin), TRKA-lineage neurons (TRKA), myelinated neurons (NEFH: the neurofilament heavy chain protein), and peptidergic neurons (CGRP: the calcitonin gene-related peptide). Bottom panels show triple staining of tdTomato with NEFH and CGRP. Arrows indicate co-localization. n = 5 mice for each marker. Data are shown as mean ± SEM. Scale bars, 100 μm.

Source data

Extended Data Fig. 2 Percentages of PROKR2ADV-tdTomato+ neurons in DRGs and their innervation in the spinal cord.

a, Representative sections through cervical (C1-C8), thoracic (T1-T13), lumbar (L1-L6), and sacral (S1-S3) DRGs from the adult Prokr2Adv-tdTomato mice. b, Percentages of DRG neurons [determined by the expression of TUBB3 (not shown), a pan-neuronal marker] expressing tdTomato along the anterior-posterior axis; n = 4 mice. Note higher representations at the limb levels (e.g., C6-C8 and L3-L6 DRGs that innervate the forelimbs and hindlimbs, respectively) compared with thoracic DRGs. c, d, Sections through L4-5 lumbar and T8-10 thoracic levels of the spinal cord from Prokr2Adv-tdTomato mice were triple stained [tdTomato (red), the NEFH protein (green) and CGRP(blue)]. tdTomato+ terminals were predominantly located in superficial laminae (c,d, top boxed panels), but also seen in deep laminae (III-V) (c,d, bottom boxed panels). Qualitatively, tdTomato+ fibers in the superficial laminae appeared to show more co-staining with CGRP and NEFH, in comparison with those in deep laminae. n = 5 mice per group. Data are shown as mean ± SEM. Scale bars, 100 μm.

Source data

Extended Data Fig. 3 Skin innervation by PROKR2ADV neurons in the hindlimb ST36 and abdominal ST25 acupoint regions.

a, Left, schematics showing the ST36 location. Middle: schematic showing of several but not all nerve bundles in the hindleg. Right: a schematic transverse section through the hindlimb ST36 region. TA: the tibialis anterior muscle. “T.”: tibia. “F.”: fibula. G.: gastrocnemius. Cpn: the common peroneal nerve and its deep (d-Cpn) and superficial (s-Cpn) branches. Lcn: the lateral cutaneous nerve. Tn: the tibial nerve. Sn: the sural nerve. b, Representative sections through the skin of the ST36 region of Prokr2Adv-tdTomato mice. tdTomato+ fibers formed circumferential endings surrounding hair follicles (“HF”), without innervation to the epidermis (“Ep.”), in contrast with epidermal presence of TUBB3+ nerve fibers (green). DAPI (blue) staining showed cell nuclei. Note that these circumferential endings coexpressed CGRP but lacked detectable expression of the NEFH protein. n = 5 mice. c, Representative skin sections through the abdominal ST25 region in Prokr2Adv-tdTomato mice. Top: tdTomato+ fibers (red) did not innervate the epidermis (“Ep.”), with DAPI (blue) staining cell nuclei. Middle and bottom: NEFH-negative but CGRP-positive tdTomato+ fibers formed circumferential endings surrounding hair follicles (“HF”). n = 5 mice. d, e, Representative sections through L4-5 lumbar DRGs (d) or through T8-10 thoracic DRGs (e) from Prokr2Adv-tdTomato mice, in which DRG neurons innervating the cutaneous tissue of ST36 and ST25 were retrogradely labeled with Fluoro-gold (blue). Note that most retrogradely labeled-PROKR2ADV-tdTomato+ neurons in both sets of DRGs coexpressed Bmpr1b mRNA (arrows) and low levels of Nefh mRNA (arrows). n = 4 mice. The bright-field images of Nefh mRNA, which were easier for identification of cells with low versus high levels, were converted to become pseudo green color in merged images. Data are shown as mean ± SEM. Scale bars, 100 μm.

Source data

Extended Data Fig. 4 Innervation patterns by PROKR2ADV neurons in deep fascial tissues at the hindlimb ST36 and abdominal ST25 regions.

a-c, tdTomato+ fibers innervated fibula periosteum (a, arrow) and the cruciate ligament (b, arrow), and tdTomato+;NEFH+ free nerve endings in the interosseous membrane located between bones (c, small arrow). tdTomato signals were, however, not detected in NEFH+ fibers (c, green, large arrow) passing through the S100+ Pacinian corpuscles (c, blue, arrowhead). n = 5 (a, b) or 2 (c) mice. d, Representative sections showing PROKR2ADV-tdTomato+ fibers that innervated tibia periosteum of the ST36 region, 63.5 ± 4.1% of which were NEFH+ and CGRP+. n = 5 mice. e, Representative transverse sections through the inner compartment of the tibialis anterior muscle (“TA”) at the ST36 region of Prokr2Adv-tdTomato mice. Most tdTomato+ fibers coexpressed NEFH (arrows), with few being NEFH-negative (arrowheads). n = 5 mice. f, g, Representative sections through L4-5 lumbar DRGs from Prokr2Adv-tdTomato mice, in which DRG neurons innervating deep ST36 tissues (deep muscles and possibly bones as well) were retrogradely labeled with Fluoro-gold (blue). Subsets of retrogradely labeled-PROKR2ADV-tdTomato+ DRG neurons expressed NefhHigh (f, arrows), NefhLow (f, arrowhead), or Bmpr1b (g, arrow). n = 4 mice. h, Representative sections showing higher innervation densities of PROKR2ADV-tdTomato+ fibers (arrows) in inner TA, compared with the outer TA muscle at the ST36 site and the abdominal muscle at the ST25 site (one-way ANOVA, n = 5 mice per group; F2, 12 = 25.098, P < 0.001; post hoc Tukey’s test: ***P < 0.001; NS = not significant, P = 0.995). i, j, Schematics showing two major subtypes of PROKR2ADV DRG neurons innervating hindlimb ST36 (i) and abdominal ST25 regions (j). NefhHigh; Bmpr1b- neurons densely innervate deep fascial tissues, including bone periosteum (“Perios.”) plus the space between muscle bundles within the inner TA compartment, but show sparse innervation in the outer TA compartment at the ST36 region and at the abdominal muscle layers of the ST25 region. The NefhLow; Bmpr1b+ neurons mainly form circumferential endings around hair follicles at both ST36 and ST25 regions, with few retrogradely labeled from deep fascia (not shown). Thoracic and lumbar DRGs also contain Nefh-negative neurons (~40%), and their target tissues remain to be determined since only a small subset (12.3%) of neurons retrogradely labeled from deep ST36 tissues were Nefh-negative. T.: tibia; F.: fibula; Ep.: epidermal; De.: dermis; Mus.: muscle; Perit.: peritoneum. Data are shown as mean ± SEM. Scale bars, 100 μm.

Source data

Extended Data Fig. 5 Intersectional genetic ablation of PROKR2ADV DRG neurons.

a, Schematics of the intersectional genetic strategy for selectively driving the diphtheria toxin receptor (“DTR”) in PROKR2Cre-marked DRG neurons that coexpressed ADVILLIN-Flpo. This was achieved upon removal of two STOP cassettes from the intersectional allele of Tau, a pan-neural gene. A Cre-dependent tdTomato allele driven from the ROSA26 promoter was included (not shown) to label all PROKR2-Cre+ cells with tdTomato, within or outside DRGs. b, Intraperitoneal injection (“i.p.”) of the diphtheria toxin (“DTX”) in Prokr2Adv-DTR mice to create PROKR2ADV-Abl mice, with littermates receiving the same DTX injections as control. c, Ablation of PROKR2Cre-tdTomato+ neurons in lumbar DRGs, as indicated by marked reduction in the percentage of TUBB3+ DRG neurons coexpressing tdTomato. n = 5 mice per group. Two-side student’s unpaired t-test, t8 = 35.61, ***P < 0.001. d, Representative images through tibia periosteum, showing reduction of TUBB3+ and NEFH+ fibers in PROKR2ADV-Abl mice compared with control mice (n = 5 mice per group; two-side student’s unpaired t-test; for TUBB3: t8 = 5.065, ***P = 0.001; for NEFH: t8 = 8.122, ***P < 0.001). e, Representative images showing the preservation of PROKR2Cre-tdTomato+ neurons in the spinal cord as well as various brain regions such as the cortex and the striatum. n = 5 mice per group. Data are shown as mean ± SEM. Scale bars, 100 μm.

Source data

Extended Data Fig. 6 PROKR2ADV neurons were dispensable for high-intensity ES of ST25 to drive sympathetic reflex and to produce anti-inflammatory effects.

a, Without LPS challenge, both control and PROKR2ADV-Abl mice showed virtually non-detectable, indistinguishable levels of TNF-α and IL-6 in serum (two-side student’s unpaired t-test, n = 5 mice per group; for TNF-α: t8 = 0.580; NS, not significant, P = 0.578; for IL-6: t8 = 0.151; NS, P = 0.884). b, Schematic description of 3.0 mA ES of the abdominal ST25 acupoint that drove spinal-sympathetic reflexes. c, No changes in 3.0 mA ST25 ES-evoked Fos (green) induction in ChAT+ preganglionic sympathetic efferent neurons in the spinal intermediolateral nuclei (“IML”) between control and PROKR2ADV-Abl mice (Two-way ANOVA, n = 5 mice per group, F1, 16 = 0.421, P = 0.562; post-hoc Tukey’s test: ***P < 0.001). d, No changes in 3.0 mA ST25 ES-evoked Fos induction in the suprarenal sympathetic ganglia (“g.”) (Two-way ANOVA, n = 5 mice per group, F1, 16 = 0.290, P = 0.598; post-hoc Tukey’s test: ***P < 0.001). e, No changes in 3.0 mA ST25 ES-induced noradrenaline (“NA”) release (two-way ANOVA, n = 6 per group, F1, 20 = 4.093, P = 0.057; post hoc Tukey test: ***P < 0.001). f, g, No changes in 3.0 mA ST25 ES-evoked reduction of LPS-induced TNF-α and IL-6 production (two-way ANOVA, n = 6 mice per group; for TNF-α: F1, 20 = 1.851, P = 0.189; post-hoc Tukey test: ***P <  < 0.001; for IL-6: F1, 20 = 5.214, P = 0.133; post-hoc Tukey test: ***P < 0.001). h, i, No marked changes in 1.0 mA ST25 ES-evoked reduction of LPS-induced TNF-α and IL-6 production (two-way ANOVA, n = 5 mice per group; for TNF-α: F1, 16 = 4.357, P = 0.053; post-hoc Tukey test: left *P = 0.014, right *P = 0.018; for IL-6: F1, 16 = 1.019, P = 0.328; post-hoc Tukey test: left *P = 0.013, right *P = 0.015). j, Schematics showing that PROKR2Cre-negative sensory neurons preserved in PROKR2ADV-Abl mice were sufficient to drive the spinal-sympathetic anti-inflammatory pathways in response to 1.0-3.0 mA ES of the abdominal ST25 acupoint. Sym. g.: sympathetic ganglia. Data are shown as mean ± SEM. Scale bars: 100 μm.

Source data

Extended Data Fig. 7 3.0 mA ES of ST36 produced anti-inflammatory effects independent of PROKR2ADV neurons.

a, Compared with control littermates, no difference of 3.0 mA ST36 ES-evoked Fos (green) induction in ChAT+ (red) sympathetic preganglionic neurons in the spinal intermediolateral nuclei (“IML”) in PROKR2ADV-Abl mice (two-way ANOVA, n = 5 mice per group, F1, 16 = 0.236, P = 0.633; post-hoc Tukey’s test: ***P < 0.001). b, No changes in 3.0 mA ST36 ES-evoked reduction of LPS-induced TNF-α and IL-6 production in PROKR2ADV-Abl mice (two-way ANOVA, n = 5 mice per group; for TNF-α: F1, 16 = 1.392, P = 0.255; post-hoc Tukey test: ***P < 0.001; for IL-6: F1, 16 = 1.382, P = 0.257; post-hoc Tukey test: ***P < 0.001). Thus, although PROKR2ADV neurons are necessary for low-intensity ES to drive the vagal-adrenal anti-inflammatory axis, they are dispensable for high-intensity ES to drive spinal-sympathetic anti-inflammatory axis from either ST25 or ST36. Data are shown as mean ± SEM. Scale bars: 100 μm.

Source data

Extended Data Fig. 8 Optogenetic activation of PROKR2ADV neurons inside the hindlimb ST36 region failed to drive sympathetic reflex.

a, Intersectional genetic strategy for generation of Prokr2Adv-CatCh (or “CatCh”) mice, in which the expression of the calcium translocating channelrhodopsin (CatCh, an L132C-mutated channelrhodopsin with enhanced Ca2+ permeability) plus the fluorescent protein EYFP was confined to PROKR2ADV DRG neurons defined by co-expression of PROKR2Cre and ADVILLINFlpo. This was achieved by crossing the intersectional CatCh mice (Ai80) with Prokr2Cre and AdvillinFlpo mice. b, c, Representative images showing that CatCh-EYFP expression was detected in a subset of L4 DRG and EYFP+ fibers innervated tibial periosteum. n = 5 mice. d, e, Electrophysiological recordings of dissociated DRG neurons from control and CatCh mice. Under the voltage clamp mode, blue light (473 nm) stimulation (10 Hz, 50 µs, 10 mW) resulted in inward currents in 26.3% (15/57) of randomly selected DRG neurons from CatCh mice (d), and after switching to the current clamp mode, this optical stimulation (10 Hz, 50 µs, 10 mW) reliably produced action potential firing (e). None of DRG neurons from control mice produced such inward currents and action potential firing (0/43 = 0%) (d, e). n = 3 mice per group. f, g, 10 mW (f) and 30 mW (g) optical stimulation of the hindlimb ST36 region in CatCh mice failed to produce an increase of Fos induction in ChAT+ preganglionic sympathetic efferent neurons located in the spinal intermediolateral nuclei (“IML”) compared to control mice (n = 5 mice per group, two-side student’s unpaired t-test; for 10 mW: t8 = 0.362; NS = not significant, P = 0.727; for 30 mW: t8 = 0.704; NS, P = 0.502). h, Schematics showing how we recorded the left cervical vagal efferent nerve in response to 473 nm optic stimulation at bilateral ST36 sites in control and CatCh mice. Note that the distal end of the vagal nerve was transected to block visceral sensory afferent inputs. Data are shown as mean ± SEM. Scale bars: 100 μm.

Source data

Extended Data Fig. 9 Optogenetic activation of hindlimb PROKR2ADV neurons induced Fos in NTS-projecting spinal neurons and in adrenal medulla-projecting DMV efferent neurons.

a, Schematics showing the experimental design for testing if optical stimulation of PROKR2ADV nerve fibers of the hindlimb ST36 region can activate spinal ascending projection neurons retrogradely labeled with Fluoro-gold from the nucleus tractus solitarius (“NTS”) in hindbrain. b, 473 nm blue light stimulation of the hindlimb ST36 region was sufficient to evoke Fos induction in NTS-projecting neurons located in the lamina I of the spinal cord in Prokr2Adv-CatCh (“CatCh”) mice (b, arrows), but not in control mice (n = 3 mice per group, two-side student’s unpaired t-test, t4 = 6.807, ***P < 0.001). This stimulation virtually did not induce any Fos in NTS-projecting neurons located in deep laminae (IV and V) of the spinal cord, in both control (1.77 ± 0.24%) and CatCh (2.27 ± 0.43%) mice (data not shown; n = 3 mice per group, two-side student’s unpaired t-test, t4 = 1.023, P = 0.364). c, Schematics showing the experimental design for testing if optical stimulation of PROKR2ADV nerve fibers of the hindlimb ST36 region can activate a subset of vagal efferent neurons located in the dorsal motor nucleus of the vagus (DMV) that were retrogradely labeled from the adrenal gland with Fluoro-gold. d, Optical stimulation of PROKR2ADV neurons of ST36 regions caused an increase in Fos induction in adrenal medulla-projecting DMV neurons compared with control mice (n = 3 mice per group, two-side student’s unpaired t-test, t4 = 8.159, ***P = 0.001). Arrows indicate retrogradely labeled DMV neurons with Fos induction. Arrowhead indicates the baseline Fos expression in ChAT-negative cells. e, No significant (NS) reduction of LPS-induced TNF-α and IL-6 production following 10 mW or 30 mW optical stimulation of PROKR2ADV fibers at the ST36 site of CatCh mice compared with control mice (two-way ANOVA, n = 5 mice per group; for TNF-α: F1, 20 = 0.124; NS, P = 0.728; post-hoc Tukey test: **P = 0.003, ***P < 0.001; for IL-6: F1, 20 = 0.714; NS, P = 0.408; post-hoc Tukey test: ***P < 0.001). Data are shown as mean ± SEM. Scale bars: 100 μm.

Source data

Extended Data Fig. 10 The tibial nerve was dispensable for focal 0.5 mA ST36 ES-evoked anti-inflammatory effects, and the common peroneal nerve was required for 0.5 mA ST36 ES to induce Fos in NTS-projecting spinal neurons.

a, 0.5 mA ST36 ES-evoked reduction of LPS-induced TNF-α and IL-6 in serum, compared with sham 0 mA ES, was unaffected by tibial nerve neurectomy (“TNX”) compared with sham surgery (“sham”) (two-way ANOVA, n = 5 mice per group; for TNF-α: F1, 16 = 0.253, P = 0.622; post hoc Tukey’s test: left **P = 0.002, right **P = 0.005; for IL-6: F1, 16 = 0.002, P =   0.989; post hoc Tukey’s test: left **P = 0.009, right **P = 0.007). As described in Extended Data Fig. 3a, the tibial nerve was located posterior to fibula and tibia, and our focal ES of ST36 apparently failed to activate this nerve. This is different from reported activation of this nerve via a diffuse ES mode10, in which the electric current entered the left ST36 site and came out of the right ST36 site10. b, 0.5 mA, but not 0 mA control, ES of ST36 induced Fos (green) in Fluoro-gold+ retrogradely labeled NTS-projecting neurons (red) located in the lamina I of the spinal cord in sham surgery mice, with arrows indicating co-labeling. This induction was lost in mice with common peroneal neurectomy (“CPX”) (Two-way ANOVA, n = 3 mice per group, F1, 8 = 265.645, P < 0.001; post-hoc Tukey’s test: ***P < 0.001; NS, not significant, P =   0.145). Data are shown as mean ± SEM. Scale bars: 100 μm.

Source data

Extended Data Fig. 11 0.5 mA ES at cutaneous or traditional non-acupoint regions failed to suppress inflammation, but ES at the forelimb LI10 acupoint can evoke PROKR2ADV neuron-dependent anti-inflammatory effects.

a, b, low-intensity ES at the superficial ST36 region. Schematics (a) showing ES at the superficial, intradermal part of the ST36 region. Two electric needles were inserted through the epidermis (“Ep.”) and into the dermis (“De.”) at ST36 regions, with needles tilted to restrict them within the superficial dermis. This intradermal 0.5-mA ES failed to reduce LPS-induced TNF-α and IL-6 expression compared with sham 0 mA ES in C57BL/6J mice (b, n = 5 mice per group; two-side student’s unpaired t-test; for TNF-α: t8 = 0.218; NS, not significant, P = 0.833; for IL-6: t8 = 0.562; NS, P = 0.589). c, d, Low-intensity stimulation of the sural nerve. Schematics (c) showing ES at the middle region of the posterior hind leg, by inserting electric needles through the Chengjin (BL56) acupoint, with tips flanking the skin-innervating sural nerve (“Sn”). 0.5 mA ES at this acupoint failed to reduce TNF-α and IL-6 compared with sham 0 mA ES in C57BL/6J mice (d, n = 5 mice per group; two-side student’s unpaired t-test; for TNF-α: t8 = 0.375; NS, P = 0.718; for IL-6: t8 = 0.721; NS, P = 0.491). e-j, Low-intensity ES within the gastrocnemius (“G.”) muscle or the semitendinosus (“S.”) muscles. Schematics (e, h) showing ES at these two muscles. Two representative images (f, i) showing sparse innervation by PROKR2ADV-tdTomato+ fibers within these muscles, with the percentages of unit areas showing positive fibers (0.51 ± 0.19% for G. and 0.60 ± 0.11% for S. muscles) comparable to that seen in the outer TA muscle (0.51 ± 0.15%) and the abdominal wall muscles (0.59 ± 0.12%) shown in Extended Data Fig. 4h. n = 4 mice. In comparison with sham 0 mA ES, no impact on TNF-α and IL-6 production by 0.5 mA ES in either G. muscle (g, n = 5 mice per group. two-side student’s unpaired t-test; for TNF-α: t8 = 0.205; NS, P = 0.843; for IL-6: t8 = 0.861; NS, P = 0.415) or in S. mucle (j, n = 5 mice per group; two-side student’s unpaired t-test; for TNF-α: t8 = 0.468; NS, P = 0.652; for IL-6: t8 = 0.593; NS, P = 0.570) in C57BL/6J mice. k-m, Low-intensity ES at the forelimb acupoint LI10 (Shousanli). Schematics (k) and the image (l, top) showing ES at the forelimb acupoint LI10. Representative images (l) showing PROKR2ADV-tdTomato+ fibers within the deep branch of the radial nerve, and their innervations in radius periosteum, which were prominent at transverse sections at levels slightly distal (e.g., 1 mm) from the LI10 acupoint level. n = 3 mice. (m) Loss of 0.5 mA LI10 ES-evoked reduction of TNF-α and IL-6 in PROKR2ADV-Abl mice compared with control mice (two-way ANOVA, n = 5 mice per group; for TNF-α: F1, 16 = 20.384, P < 0.001; post-hoc Tukey test: *P = 0.011; NS, P = 0.562; for IL-6: F1, 16 = 14.296, P = 0.002; post-hoc Tukey test: **P = 0.004; NS, P = 0.728). (n) Loss of 0.5 mA LI10 ES-evoked reduction of TNF-α and IL-6 in mice with subdiaphragmatic vagotomy (“sVX”) compared with mice with sham surgery (two-way ANOVA, n = 5 mice per group; for TNF-α: F1, 16 = 22.875, P < 0.001; post-hoc Tukey test: **P = 0.004; NS, P = 0.697; for IL-6: F1, 16 = 18.065, P = 0.002; post-hoc Tukey test: **P = 0.004; NS, P = 0.57). TA: Anterior tibial muscle; T.: Tibia; F.: Fibula; Fe.: Femur; R.: Radius; U.: Ulna. Data are shown as mean ± SEM. Scale bars: 100 μm.

Source data

Supplementary information

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, S., Wang, Z., Su, Y. et al. A neuroanatomical basis for electroacupuncture to drive the vagal–adrenal axis. Nature 598, 641–645 (2021). https://doi.org/10.1038/s41586-021-04001-4

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links