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 sites1-6 (for example, suppressing severe systemic inflammation6-9). Since the 1970s, an emerging organizational rule about these reflexes has been the presence of body-region specificity1-6. 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.

© 2021. The Author(s), under exclusive licence to Springer Nature Limited.

Figures

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.

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.

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.

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. n = 5 mice. f, g, Representative sections through L4-L5 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 outer TA and abdominal muscles (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.

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 receive 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.

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-a and IL-6 in serum (two-side student’s unpaired t-test, n = 5 mice per group; for TNF-a: 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-a and IL-6 production (two-way ANOVA, n = 6 mice per group; for TNF-a: 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-a and IL-6 production (two-way ANOVA, n = 5 mice per group; for TNF-a: 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.

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-a and IL-6 production in PROKR2ADV-Abl mice (two-way ANOVA, n = 5 mice per group; for TNF-a: 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.

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.

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-a 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-a: 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.

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-a 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 mode, in which the electric current entered the left ST36 site and came out of the right ST36 site. 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.

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-a 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-a: 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-a 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-a: 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-a 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-a: 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-a: 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-a and IL-6 in PROKR2ADV-Abl mice compared with control mice (two-way ANOVA, n = 5 mice per group; for TNF-a: 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-a 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-a: 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.

Fig. 1:. Characterizing PROKR2ADV neurons.

a, Schematic of the somatotopic organization that drives two autonomic pathways by ES. Sym., sympathetic. b, Sections through L4–L5 and T8–T10 DRGs from Prokr2Adv-tdTomato mice showing the colocalization of tdTomato (red) with Nefh mRNA expression (bright-field images converted to pseudo-green in merged images) and the pan-neuronal marker TUBB3 (blue). Arrows and arrowheads indicate tdTomato+ neurons with high and low levels of Nefh mRNA expression, respectively. n = 4 mice. c, The percentage of TUBB3+ neurons expressing the indicated markers in L4–L5 and T8–T10 DRGs (n = 4 mice; two-sided Student’s unpaired t-test; for tdTomato+, t6 = 7.512, ***P < 0.001; for tdTomato+Nefhhigh,t6 = 7.201, ***P < 0.001; for tdTomato+Nefhlow, t6 = 1.714, not significant (NS), P = 0.137). d, e, Transverse and sagittal sections through the tibial bone periosteum (d), and transverse sections plus whole-mount preparation of the abdominal peritoneum (e) from adult Prokr2Adv-tdTomato mice showing tdTomato (red) and TUBB3 (green) localization. n = 5 mice. Scale bars, 100 μm. Data are mean ± s.e.m.

Fig. 2:. Requirement of PROKR2ADV neurons for low-intensity ES to drive the vagal–adrenal anti-inflammatory axis.

a, Schematic of ES at the ST36 site. The red and green dots close to the needle tips represent the deep and superficial branches, respectively, of the peroneal nerve. F, fibula; T, tibia. b, Images (left) and quantification (right) of Fos expression (green) induced by ES (0.5 mA) at the ST36 acupoint in ChAT+ (red) DMV neurons in control but not in PROKR2ADV-Abl mice. Two-way ANOVA, n = 5 mice, F1, 16 = 26.769, P < 0.001; post hoc Tukey’s test: ***P < 0.001; NS, P = 0.204. Arrow, ChAT+ cells in DMV; arrowheads, Fos+ChAT+ neurons. c, ES (0.5 mA) at the ST36 site increased the release of noradrenaline (NA), adrenaline (A) and dopamine (DA) in control but not PROKR2ADV-Abl mice. Two-way ANOVA, n = 6 mice, F1, 20 = 11.603 (NA), 21.338 (A), 33.220 (DA), P < 0.001; post hoc Tukey test: ***P < 0.001; NS, P = 0.535 (NA), 0.487 (A) and 0.450 (DA). d, e, PROKR2ADV-Abl mice exhibited loss of 0.5-mA ES ST36-evoked TNF (d) and IL-6 (e) anti-inflammatory effects compared with control mice. Two-way ANOVA, n = 6 mice; for TNF: F1, 20 = 40.025, P < 0.001; post hoc Tukey test: **P = 0.003; NS, P = 0.596; for IL-6: F1, 20 = 28.573, P < 0.001; post hoc Tukey test: **P = 0.002; NS, P = 0.931. f, g, ES (0.5 mA) at the ST36 site (compared with 0 mA ES) increased survival rates in LPS-treated control mice (f; two-sided log-rank test, **P = 0.008) but not in PROKR2ADV-Abl mice (g; two-sided log-rank test, NS, P = 0.456). n = 36 (0 mA) or 35 (0.5 mA) mice. Scale bars, 100 μm. Data are mean ± s.e.m.

Fig. 3:. Activation of PROKR2ADV fibres drives the vagal–adrenal anti-inflammatory axis.

a, Schematic of optical stimulation of the ST36 acupoint in control and Prokr2Adv-CatCh (CatCh) mice. Perios, periosteum. b, Images (left) and quantification (right) of Fos (green) induction in ChAT+ (red) DMV neurons (n = 5 mice; two-sided Student’s unpaired t-test, t8 = 8.713, ***P < 0.001). Arrow, ChAT+ cells in DMV; arrowheads, Fos+ChAT+ neurons. c, Left, example vagal efferent electrical activity traces from CatCh and control mice. Right, CatCh mice exhibit increased firing compared with control mice (n = 5 mice per group; two-sided Student’s unpaired t-test, t8 = 4.855, **P = 0.001). d,CatCh mice show loss of increased release of noradrenaline, adrenaline and dopamine induced by optical activation of PROKR2ADV fibres following subdiaphragmatic vagotomy (sVX) compared with mice that underwent sham surgery. Two-way ANOVA, n = 6 mice, F1, 20 = 33.278 (NA), 33.188 (A), 80.020 (DA), P < 0.001; post hoc Tukey’s paired comparisons: ***P < 0.001; NS, P = 0.326 (NA), P = 0.584 (A) and P = 0.697 (DA). – denotes control littermates and + denotes CatCh mice. e, f, Optical activation of PROKR2ADV fibres reduced LPS-induced production of TNF (e) and IL-6 (f) in mice that underwent sham surgery but not in sVX mice. Two-way ANOVA, n = 6 mice; for TNF: F1, 20 = 8.021, P < 0.001; for IL-6: F1, 20 = 10.695, P = 0.004; post hoc Tukey’s test: **P = 0.003 (TNF) or P = 0.001 (IL-6); NS, P = 0.535 (TNF) or P = 0.486 (IL-6). g, Optical activation of PROKR2ADV fibres promoted survival in CatCh mice (n = 22 mice per group, two-sided log-rank test, ***P < 0.001). Scale bars, 100 μm. Data are mean ± s.e.m.

Fig. 4:. Requirement of deep-tissue-innervating nerves for anti-inflammatory effects.

a, Schematic of common peroneal nerve (Cpn) neurectomy (CPX) and lateral cutaneous nerve (Lcn) neurectomy (CLX). b, CPX mice showed preservation of TUBB3+ fibres (green) in the epidermis (Ep.) and dermis (De.) of skin at the ST36 region (top), but loss in the tibial periosteum (the anterior side; bottom). HF, hair follicle. n = 3 mice. c, ES (0.5 mA) at ST36 reduced LPS-induced production of TNF (left) and IL-6 (right) in mice that underwent sham surgery compared with CPX. Two-way ANOVA, n = 5 mice; for TNF, F1, 16 = 13.653, P = 0.002; for IL-6: F1, 16 = 6.917, P = 0.018; post hoc Tukey’s test: ***P < 0.001; NS, P = 0.652 for TNF and P = 0.687 for IL-6. d, CLX mice showed loss of TUBB3+ nerve fibres (upper panel) in the skin at the ST36 site (top), but not in tibial periosteum (bottom). n = 3 mice. e, There were no changes in ES-evoked reduction of LPS-induced production of TNF (left) and IL-6 (right) in mice that underwent CLX compared with sham surgery. Two-way ANOVA, n = 5 mice; for TNF: F1, 16 = 0.843, P = 0.372; for IL-6: F1, 16 = 1.229, P = 0.284; post hoc Tukey’s test: **P = 0.001, ***P < 0.001. f, Schematic of the vagal–adrenal axis driven by PROKR2Cre neurons innervating deep limb fascial tissues. Scale bars, 100 μm. Data are mean ± s.e.m.