Animals and humans display two types of response to noxious stimuli. The first includes reflexive defensive responses that prevent or limit injury; a well-known example of these responses is the quick withdrawal of one’s hand upon touching a hot object. When the first-line response fails to prevent tissue damage (for example, a finger is burnt), the resulting pain invokes a second-line coping response—such as licking the injured area to soothe suffering. However, the underlying neural circuits that drive these two strings of behaviour remain poorly understood. Here we show in mice that spinal neurons marked by coexpression of TAC1Cre and LBX1Flpo drive coping responses associated with pain. Ablation of these spinal neurons led to the loss of both persistent licking and conditioned aversion evoked by stimuli (including skin pinching and burn injury) that—in humans—produce sustained pain, without affecting any of the reflexive defensive reactions that we tested. This selective indifference to sustained pain resembles the phenotype seen in humans with lesions of medial thalamic nuclei1,2,3. Consistently, spinal TAC1-lineage neurons are connected to medial thalamic nuclei by direct projections and via indirect routes through the superior lateral parabrachial nuclei. Furthermore, the anatomical and functional segregation observed at the spinal level also applies to primary sensory neurons. For example, in response to noxious mechanical stimuli, MRGPRD- and TRPV1-positive nociceptors are required to elicit reflexive and coping responses, respectively. Our study therefore reveals a fundamental subdivision within the cutaneous somatosensory system, and challenges the validity of using reflexive defensive responses to measure sustained pain.
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All data are contained in the main text or Supplementary Materials or are available from the corresponding author upon reasonable request. The anterograde tracing data are from the Allen Brain Atlas website (http://connectivity.brain-map.org/).
Mark, V. H., Ervin, F. R. & Yakovlev, P. I. Stereotactic thalamotomy III. The verification of anatomical lesion sites in the human thalamus. Arch. Neurol. 8, 528–538 (1963).
Young, R. F. et al. Gamma knife thalamotomy for the treatment of persistent pain. Stereotact. Funct. Neurosurg. 64, 172–181 (1995).
Price, D. D. Central neural mechanisms that interrelate sensory and affective dimensions of pain. Mol. Interv. 2, 392–403, 339 (2002).
Gutierrez-Mecinas, M. et al. Preprotachykinin A is expressed by a distinct population of excitatory neurons in the mouse superficial spinal dorsal horn including cells that respond to noxious and pruritic stimuli. Pain 158, 440–456 (2017).
Todd, A. J. Neuronal circuitry for pain processing in the dorsal horn. Nat. Rev. Neurosci. 11, 823–836 (2010).
Bourane, S. et al. Identification of a spinal circuit for light touch and fine motor control. Cell 160, 503–515 (2015).
Head, H. & Holmes, H. J. Sensory disturbances from cerebral lesions. Brain 34, 102–154 (1911).
Han, S., Soleiman, M. T., Soden, M. E., Zweifel, L. S. & Palmiter, R. D. Elucidating an affective pain circuit that creates a threat memory. Cell 162, 363–374 (2015).
Rodriguez, E. et al. A cranio-facial-specific monosynaptic circuit enables hieghtened affective pain. Nat. Neurosci. 20, 1734–1743 (2017).
Yahiro, T., Kataoka, N., Nakamura, Y. & Nakamura, K. The lateral parabrachial nucleus, but not the thalamus, mediates thermosensory pathways for behavioural thermoregulation. Sci. Rep. 7, 5031 (2017).
Mu, D. et al. A central neural circuit for itch sensation. Science 357, 695–699 (2017).
Oh, S. W. et al. A mesoscale connectome of the mouse brain. Nature 508, 207–214 (2014).
Duan, B. et al. Identification of spinal circuits transmitting and gating mechanical pain. Cell 159, 1417–1432 (2014).
Bourane, S. et al. Gate control of mechanical itch by a subpopulation of spinal cord interneurons. Science 350, 550–554 (2015).
Melzack, R. & Wall, P. D. Pain mechanisms: a new theory. Science 150, 971–979 (1965).
Duan, B., Cheng, L. & Ma, Q. Spinal circuits transmitting mechanical pain and itch. Neurosci. Bull. 34, 186–193 (2018).
Arenas, O. M. et al. Activation of planarian TRPA1 by reactive oxygen species reveals a conserved mechanism for animal nociception. Nat. Neurosci. 20, 1686–1693 (2017).
Vandewauw, I. et al. A TRP channel trio mediates acute noxious heat sensing. Nature 555, 662–666 (2018).
Ward, L., Wright, E. & McMahon, S. B. A comparison of the effects of noxious and innocuous counterstimuli on experimentally induced itch and pain. Pain 64, 129–138 (1996).
Cavanaugh, D. J. et al. Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli. Proc. Natl Acad. Sci. USA 106, 9075–9080 (2009).
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).
Cavanaugh, D. J. et al. Restriction of transient receptor potential vanilloid-1 to the peptidergic subset of primary afferent neurons follows its developmental downregulation in nonpeptidergic neurons. J. Neurosci. 31, 10119–10127 (2011).
Malin, S. et al. TRPV1 and TRPA1 function and modulation are target tissue dependent. J. Neurosci. 31, 10516–10528 (2011).
Cheng, L. et al. Identification of spinal circuits involved in touch-evoked dynamic mechanical pain. Nat. Neurosci. 20, 804–814 (2017).
Adriaensen, H., Gybels, J., Handwerker, H. O. & Van Hees, J. Nociceptor discharges and sensations due to prolonged noxious mechanical stimulation—a paradox. Hum. Neurobiol. 3, 53–58 (1984).
Schmidt, R., Schmelz, M., Torebjörk, H. E. & Handwerker, H. O. Mechano-insensitive nociceptors encode pain evoked by tonic pressure to human skin. Neuroscience 98, 793–800 (2000).
Schmelz, M., Schmid, R., Handwerker, H. O. & Torebjörk, H. E. Encoding of burning pain from capsaicin-treated human skin in two categories of unmyelinated nerve fibres. Brain 123, 560–571 (2000).
Cobos, E. J. & Portillo-Salido, E. ‘Bedside-to-bench’ behavioral outcomes in animal models of pain: beyond the evaluation of reflexes. Curr. Neuropharmacol. 11, 560–591 (2013).
Borsook, D., Hargreaves, R., Bountra, C. & Porreca, F. Lost but making progress—where will new analgesic drugs come from? Sci. Transl. Med. 6, 249sr3 (2014).
LeDoux, J. E. & Pine, D. S. Using neuroscience to help understand fear and anxiety: a two-system framework. Am. J. Psychiatry 173, 1083–1093 (2016).
Niederkofler, V. et al. Identification of serotonergic neuronal modules that affect aggressive behavior. Cell Rep. 17, 1934–1949 (2016).
Britz, O. et al. A genetically defined asymmetry underlies the inhibitory control of flexor–extensor locomotor movements. eLife 4, e04718 (2015).
Liu, Y. et al. VGLUT2-dependent glutamate release from nociceptors is required to sense pain and suppress itch. Neuron 68, 543–556 (2010).
Lou, S., Duan, B., Vong, L., Lowell, B. B. & Ma, Q. Runx1 controls terminal morphology and mechanosensitivity of VGLUT3-expressing C-mechanoreceptors. J. Neurosci. 33, 870–882 (2013).
Knowlton, W. M., Bifolck-Fisher, A., Bautista, D. M. & McKemy, D. D. TRPM8, but not TRPA1, is required for neural and behavioral responses to acute noxious cold temperatures and cold-mimetics in vivo. Pain 150, 340–350 (2010).
Brenner, D. S., Golden, J. P. & Gereau, R. W., IV. A novel behavioral assay for measuring cold sensation in mice. PLoS ONE 7, e39765 (2012).
Pogorzala, L. A., Mishra, S. K. & Hoon, M. A. The cellular code for mammalian thermosensation. J. Neurosci. 33, 5533–5541 (2013).
Green, B. G. et al. Evaluating the ‘Labeled Magnitude Scale’ for measuring sensations of taste and smell. Chem. Senses 21, 323–334 (1996).
Bartoshuk, L. M. et al. Valid across-group comparisons with labeled scales: the gLMS versus magnitude matching. Physiol. Behav. 82, 109–114 (2004).
Green, B. G. & Schoen, K. L. Thermal and nociceptive sensations from menthol and their suppression by dynamic contact. Behav. Brain Res. 176, 284–291 (2007).
LaMotte, R. H., Shimada, S. G., Green, B. G. & Zelterman, D. Pruritic and nociceptive sensations and dysesthesias from a spicule of cowhage. J. Neurophysiol. 101, 1430–1443 (2009).
Sikand, P., Shimada, S. G., Green, B. G. & LaMotte, R. H. Sensory responses to injection and punctate application of capsaicin and histamine to the skin. Pain 152, 2485–2494 (2011).
Kleinlogel, S. et al. Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh. Nat. Neurosci. 14, 513–518 (2011).
Kwan, K. Y. et al. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction. Neuron 50, 277–289 (2006).
Story, G. M. et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112, 819–829 (2003).
Bautista, D. M. et al. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 124, 1269–1282 (2006).
Wang, S. et al. Phospholipase C and protein kinase A mediate bradykinin sensitization of TRPA1: a molecular mechanism of inflammatory pain. Brain 131, 1241–1251 (2008).
Bartley, E. J. & Fillingim, R. B. Sex differences in pain: a brief review of clinical and experimental findings. Br. J. Anaesth. 111, 52–58 (2013).
Doehring, A. et al. Effect sizes in experimental pain produced by gender, genetic variants and sensitization procedures. PLoS ONE 6, e17724 (2011).
Giesler, G. J. Jr, Yezierski, R. P., Gerhart, K. D. & Willis, W. D. Spinothalamic tract neurons that project to medial and/or lateral thalamic nuclei: evidence for a physiologically novel population of spinal cord neurons. J. Neurophysiol. 46, 1285–1308 (1981).
Benabid, A. L. & Jeaugey, L. Cells of the rat lateral habenula respond to high-threshold somatosensory inputs. Neurosci. Lett. 96, 289–294 (1989).
Abdel Samad, O. et al. Characterization of two Runx1-dependent nociceptor differentiation programs necessary for inflammatory versus neuropathic pain. Mol. Pain 6, 45 (2010).
Kiasalari, Z. et al. Identification of perineal sensory neurons activated by innocuous heat. J. Comp. Neurol. 518, 137–162 (2010).
Chen, C. L. et al. Runx1 determines nociceptive sensory neuron phenotype and is required for thermal and neuropathic pain. Neuron 49, 365–377 (2006).
We thank Z. J. Huang, M. J. Zylka, M. Hoon, S. M. Dymecki and the Allen Brain Institute/the Jackson Laboratory for genetically modified mice; A. I. Basbaum, A. V. Apkarian and C. J. Woolf for constructive discussions; and Y. Liu and Z. He for viral reagents. The work was supported by National Institutes of Health grants to Q.M. (R01 DE018025) and to Q.M. and M.G. (R01 NS086372), a Wellcome Trust grant to Q.M. (200183/Z/15/Z), and a Research Fellowship (326726541) from the German Research Foundation (DFG) to N.M.M.
Nature thanks F. Wang and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a–c, Lumbar spinal sections from P30 Tac1cre-tdTomato mice (n = 3), in which spinal neurons with developmental expression of TAC1Cre were labelled by tdTomato, showing double staining of tdTomato signals (red) and the mRNA of the excitatory neuronal marker VGLUT2 (a, green), or inhibitory neuronal markers GAD67 (b, green) and GlyT2 (c, green) detected by in situ hybridization13. Right panels represent a higher magnification of the boxed areas. Arrows show co-localization, and arrowheads show singular expression. Quantification of neurotransmitter phenotypes of spinal TAC1Cre–tdTomato+ neurons: 91.2 ± 0.7% (± s.e.m.) are VGLUT2+ excitatory neurons, 6.1 ± 1.1% (± s.e.m.) are GAD67+ GABAergic inhibitory neurons and 4.5 ± 0.9% (± s.e.m.) are GlyT2+ glycinergic inhibitory neurons. d, Intersectional genetic strategy for driving tdTomato expression in spinal TAC1CDX2 neurons defined by co-expression of TAC1Cre and CDX2Flpo. It had previously been reported that CDX2Flpo drives reporter expression from the cervical spinal cord all the way to the most-caudal spinal cord6. By crossing Tac1cre mice and Cdx2Flpo mice with intersectional Ai65 reporter mice, only spinal neurons with developmental co-expression of TAC1Cre and CDX2Flpo drove tdTomato expression; these are referred to as Tac1Cdx2-tdTomato mice. e, Representative sections through the spinal cord of Tac1Cdx2-tdTomato mice (n = 3), showing that tdTomato+ neurons are not detected in the most-rostral cervical levels or in the brain (data not shown), but are detected in the lumbar levels. f, Representative coronal sections (25-μm thick, prepared by cryostat; in comparison with Fig. 1c, which was 100-μm thick, and prepared by vibratome) through the ventral lateral thalamus of Cdx2Flpo-tdTomato mice (left, n = 3) and Tac1Cdx2-tdTomato mice (right, n = 3). Cdx2Flpo-tdTomato mice were generated by crossing Cdx2Flpo mice with Flpo-dependent Rosa26FSF-tdTomato reporter mice. Among 25-μm-thick sections of VPL from Tac1Cdx2-tdTomato mice, only 12% (3 out of 26) showed sparse tdTomato signals, whereas 100% (26 out of 26) of sections from Cdx2Flpo-tdTomato mice showed robust tdTomato signals (χ2 test, χ20.95,(1) = 41.241, P < 0.001). It should be noted that owing to the restriction of CDX2Flpo to the spinal cord, no tdTomato signals were detected in VPM of Cdx2Flpo-tdTomato mice, because VPM are innervated by neurons located in the trigeminal nuclei or dorsal column nuclei that were not labelled by CDX2Flpo. g, h, Representative coronal sections (100-μm thick) through the thalamus of P30 Cdx2Flpo-tdTomato mice (n = 2) at the level of bregma −1.70 mm, showing whole spinal ascending fibres in the medial thalamic complex. i, Representative coronal sections (25 μm) of PBN from P30 Cdx2Flpo-tdTomato mice (n = 2), showing tdTomato (red) and CGRP immunostaining (green). CDX2Flpo–tdTomato+ fibres send collateral terminals to CGRP+ PBel as indicated by the arrow, besides projections to PBel and PBdvl. Scale bars, 50 μm (a–c), 100 μm (e–i). Source Data
a, Representative images showing the distribution of presynaptic reporter (the synaptophysin–EGFP fusion protein)12 in the dorsal portion of lateral PBN—including PBsl (a, middle, green)—but not in the more ventral PBel, following intraspinal injection of the AAV-Syn1-DIO-tdTomato-T2A-SynEGFP virus at the lumbar level of adult Tac1cre mice (n = 2). The TAC1Cre+ axons are visualized by tdTomato signals (red). Arrowhead indicates potential axons that pass through ventral lateral PBN without making synapses. b, Intersectional genetic strategy for driving the expression of the calcium translocating channelrhodopsin (CatCh, an L132C mutant channelrhodopsin with enhanced Ca2+ permeability and fused with GFP)43 in spinal TAC1CDX2 neurons defined by co-expression of TAC1Cre and CDX2Flpo. This was achieved by crossing the intersectional CatCh mice (Ai80) with Tac1cre and Cdx2Flpo, with the resulting triple heterozygous mice referred to as Tac1Cdx2-CatCh mice. Triple heterozygous Tac1Cdx2-GFP mice—generated by crossing Tac1cre and Cdx2Flpo mice with intersectional GFP reporter mice RC:: FrePe—were used as control. c, The ascending TAC1CDX2–CatCh–GFP+ terminals (observed from 3 mice) were detected in the medial thalamic region (left), including the PVT and MTh. Right, GFP signals in PBsl. The fluorescent signal of CatCh–GFP fusion protein detected by immunostaining is not as robust as that shown by the direct visualization of tdTomato signals observed in Tac1Cdx2-tdTomato mice, in Fig. 1. Scale bars, 100 μm. d, Top, schematic (left) of optogenetic activation of TAC1CDX2–CatCh+ terminals in PBN via 473-nm blue light, and recording sites for neurons in PBsl or PBel (middle and right, respectively) of the same brain slices. Bottom, voltage clamp (V-clamp) was used to record the evoked excitatory postsynaptic currents (EPSC), with holding membrane potential (HP) at −70 mV. Current clamp was used to record action potential (AP) firing. RP, resting membrane potential. Representative recording traces show that neurons in PBsl but not in PBel responded to the blue-light stimulation (0.2 Hz, 20 ms, numbers of neurons with responses: PBsl, 4 out of 15; PBel, 0 out of 15; χ2 test, χ20.95,(1) = 4.615, P = 0.032; Tac1Cdx2-CatCh mice, n = 2). Source Data
Extended Data Fig. 3 Retrograde labelling of spinal Tac1cre-marked projection neurons from parabrachial and medial thalamic nuclei, and anterograde tracing from the dorsal part of lateral parabrachial nuclei.
a, Fluorogold retrograde labelling from PBN of Tac1cre-tdTomato mice (n = 3). Left, injection site. Middle and right, representative transverse section of the dorsal horn showing fluorogold+ retrograde-labelled cells (green) and tdTomato+ TAC1-lineage neurons (red). Arrows indicate colocalization in the lateral spinal nucleus (a1) and deep laminae (a2). Arrowhead indicates a tdTomato-negative retrograde labelled neuron, showing that TAC1Cre-marked neurons represent a subset of spinoparabrachial projection neurons (n = 3, 27.2 ± 0.7%). b, Fluorogold retrograde labelling from the medial thalamic nuclei (n = 3 mice). Left, injection site. Large arrowhead indicates that the fluorogold injection did not leak to the lateral VPM–VPL complex. Middle and right, a representative transverse section of the dorsal horn, showing fluorogold+ retrograde-labelled cells (green) and tdTomato+ Tac1-lineage neurons (red). Arrows indicate colocalization in the lateral spinal nucleus (b1) and deep laminae (b2). Small arrowheads indicate tdTomato-negative retrograde-labelled neurons, indicating that TAC1Cre-marked neurons again represent a subset of spinothalamic projection neurons (n = 3 mice, 16.3 ± 3.8%). c, Anterograde tracing from dorsal lateral PBN (including PBsl and PBdvl). Image credit, Allen Institute. Left, the coronal plane (bregma −5.10 mm) showing the injection site in parabrachial nuclei. Arrow indicates tracer injection confined to PBsl plus PBdvl. The white-dotted circle (arrowhead) indicates the PBel that contains little or no injected tracer. Right, the projection of neurons from PBsl and PBdvl to thalamic and hypothalamic regions (bregma −1.70 mm). Boxes d1, d2 and d3 highlight projections or lack of projections to medial thalamic nuclei, ventral lateral thalamic nuclei and amygdaloid nuclei shown in d, respectively. HY, hypothalamic nuclei. d, Dense innervations were observed in medial thalamic nucleus, lateral habenular nuclei and the paraventricular nucleus of the thalamus (d1). No innervations were observed in VPM or VPL (d2), or the central (CeA) and basal lateral (BLA) parts of amygdala (d3). The lack of innervations to CeA, which is innervated by CGRP+ neurons in PBel8, provided a further indication that the tracer injection to the PBsl–PBdvl region did not diffuse to the PBel region. The full set of tracing images is available at the Allen Mouse Brain Connectivity Atlas12 (http://connectivity.brain-map.org/projection/experiment/siv/127469566?imageId=127469776&imageType=TWO_PHOTON,SEGMENTATION&initImage=TWO_PHOTON&x=18728&y=17591&z=3; injection site picture, modified from image 104 of 140; thalamic projection picture, modified from image 71 of 140). Source Data
Extended Data Fig. 4 Additional anatomical and behavioural characterizations of mice in which TAC1LBX1 neurons were ablated, as well as temporal segregation of withdrawal versus licking responses evoked by noxious heat and their correlation with Fos induction in TAC1Cre-marked neurons.
a, Intersectional genetic strategy for driving DTR expression selectively in dorsal spinal cord TAC1LBX1 neurons defined by co-expression of TAC1Cre and LBX1Flpo. DTR is driven from the pan-neural promoter Tau, and its expression requires removal of two STOP cassettes by Cre and Flpo DNA recombinases. LBX1Flpo expression is confined to the dorsal hindbrain and dorsal spinal cord within the nervous system6,13. b, Representative images showing a marked loss of tdTomato+ cells in the hindbrain spinal trigeminal nucleus (SpV) after diphtheria toxin injections (n = 3 mice). Arrow in SpV indicates one of few remaining cells; arrowhead indicates processes derived from TAC1Cre–tdTomato+ trigeminal primary afferents that were preserved. TAC1Cre–tdTomato+ neurons are preserved in dorsal root ganglia (DRG) and trigeminal ganglia (not shown), as well as in various brain regions such as the cortex, hippocampus formation (HPF), periaqueductal grey nuclei (PAG) or raphe magnus. Aq, aqueduct. c, No detected difference in falling latencies from the rotarod between control littermates and TAC1LBX1-Abl mice (control, n = 13 mice; TAC1LBX1-Abl, n = 14 mice; two-sided t-test, P = 0.403). d, No detected difference in response rates to gentle hindpaw brushing (out of three tries for each mouse) between control and TAC1LBX1-Abl groups (control, n = 13 mice; TAC1LBX1-Abl, n = 14 mice; two-sided Mann–Whitney rank-sum test, P = 0.121). e, Wild-type mice showed distinct latencies of lifting or flinching versus licking in response to hot plate stimulation set at 46–47 °C, but no difference at 50 °C (46 °C, n = 10 mice, two-sided paired t-test, P = 0.006; 47 °C, n = 10 mice, two-sided paired t-test, P < 0.001; 50 °C, n = 12 mice, two-sided paired t-test, P = 0.379). In the 46-°C hot plate test, licking responses were rarely observed within the first 3 min. f, Representative immunostaining of Fos in superficial dorsal horn of Tac1cre-tdTomato mice 2 h after 3-min exposure to the 46-°C or 50-°C hot plate (n = 3 mice for each condition). Only 50 °C could induce robust Fos expression. Bottom panels represent the boxed area shown above. Arrows indicate colocalization of Fos (green) with TAC1Cre-marked tdTomato+ cells (red). Scale bars, 100 μm (b), 25 μm (f). Data are presented as mean ± s.e.m. (c, e) or median ± quartile (d). Source Data
Extended Data Fig. 5 Mice in which TAC1LBX1 neurons were ablated still produced licking responses to intraplantar capsaicin injection.
No difference in licking evoked by 10 μl of a solution that contained 3 μg or 0.03 μg capsaicin (3-μg test, control, n = 15 mice; TAC1LBX1-Abl, n = 10 mice, two-sided t-test, t(23) = 1.714, P = 0.143; 0.03-μg test, control, n = 7 mice; TAC1LBX1-Abl, n = 7 mice, two-sided t-test, t(12) = 0.519, P = 0.613), suggesting the existence of pain pathways that are independent of TAC1LBX1 neurons. This preservation of capsaicin-evoked licking is markedly different from a complete loss of licking evoked by mustard oil and other noxious stimuli (Fig. 3). Licking responses evoked by mustard oil at a low concentration (≤0.75%) are dependent on TRPA144, and TRPA1 is expressed in a subset of TRPV1+ neurons45. As such, neurons that are responsive to mustard oil represent only a subset of capsaicin-responsive neurons46,47. In other words, there are neurons that are sensitive to capsaicin and insensitive to mustard oil that could—in principle—mediate licking that is independent of TAC1LBX1 neurons, and which is evoked by capsaicin. Data shown as mean ± s.e.m. Source Data
During the application of the alligator clip, both female and male subjects were instructed to rate continuously the perceived intensity of pain, regardless of its quality. After the clip was removed, each subject was asked to rate—in similar fashion—the maximal perceived intensity of each of four aversive qualities of cutaneous sensation associated with the pain they had just experienced. The four sensory qualities were itch, pricking or stinging, burning, and aching. Then, subjects were asked to rate the discomfort associated with this maximal sensation. The common scale at the right side indicates the intensity of each sensation (see Methods for detail). a, No differences between male (n = 13) and female (n = 12) human subjects in rating the magnitude of the indicated sensory qualities (two-sided Mann–Whitney rank-sum test; itch, U = 75.0, P = 0.874; pricking or stinging, U = 69.5, P = 0.663; burning, U = 71.0, P = 0.723; and aching, U = 62.5, P = 0.414; two-sided t-test, discomfort, t(23) = −0.150, P = 0.882; data shown as mean ± s.e.m.). b, No differences in the continuous pain rating between males (n = 13) and females (n = 12) (top panels, continuous pain rating at different time points during the one-minute pinch period were subjected to two-way ANOVA analyses with repeated measures, and no significant difference was detected between genders, F(1,23) = 0.008, P = 0.929; bottom, the areas under the entire curve (AUC) did not show a difference between genders, two-sided t-test, t(23) = 0.089, P = 0.929, data shown as mean ± s.e.m.). This lack of any detectable gender differences with the current sample sizes is consistent with previous studies that show that gender differences for experimentally evoked pain are not easy to detect in humans48,49. Source Data
Extended Data Fig. 7 Loss of pinch-induced Fos expression in the dorsal horn, PBsl and LHb, and attenuated pruritogen-induced scratching in mice in which TAC1LBX1 neurons were ablated.
a, Representative lumbar spinal cord sections of P60 Tac1cre-tdTomato mice (n = 4) after hindpaw pinch stimulation. We found that 41.7 ± 8.0% neurons with pinch-induced Fos co-expressed tdTomato. Arrows indicate co-localization and arrowheads indicate singular expression. b, Reduced Fos in lumbar dorsal horn of TAC1LBX1-Abl mice (n = 3 mice for each group, two-sided t-test, P = 0.007). c, Representative images showing pinch-induced Fos on coronal sections through the lateral PBN. Note that in wild-type littermates, pinch-induced Fos was enriched in PBsl, and only rarely in PBel. Right, quantification of Fos+ cells between bregma −5.24 and −4.96 mm, with and without pinching (no-pinch group, control littermates, n = 7; TAC1LBX1-Abl, n = 4 mice; pinch group, control littermates, n = 8; TAC1LBX1-Abl, n = 7 mice). Two-way ANOVA indicates significant interactions between genotypes and pinch stimulation (F(1,22) = 8.555, P = 0.008); post hoc comparison (Holm–Sidak method) shows comparable basal levels of Fos expression (no-pinch groups, P = 0.72), an increase in control littermates within PBsl (P = 0.004) and the loss of this increase in TAC1LBX1-Abl mice (P = 0.006). d, Representative coronal sections through the dorsal midline thalamic complex, showing bilateral Fos induction by pinch, which is consistent with previous electrophysiological studies50,51. Right, counting of pinch-induced Fos+ cells in a region of LHb that is adjacent to MHb from bregma −1.46 to −2.06 mm (no-pinch groups, control littermates, n = 4; TAC1LBX1-Abl, n = 4 mice; pinch groups, control littermates, n = 7; TAC1LBX1-Abl, n = 7 mice). Two-way ANOVA indicates significant interactions between genotypes and pinch stimulation (F(1,18) = 11.08, P = 0.004); post hoc comparison (Holm–Sidak method) shows comparable basal-level Fos expression (no-pinch groups, P = 0.289), significant increase in LHb of control littermates (P = 0.008) and loss of this increase in TAC1LBX1-Abl mice (P = 0.003). Owing to high background expression of Fos in the PVT and MTh, we cannot determine pinch-evoked neuronal activation in these nuclei. e, No difference in scratching response rates evoked by light von-Frey-filament stimulation (control, n = 8 mice; TAC1LBX1-Abl, n = 8 mice; two-sided Mann–Whitney rank-sum test, P = 0.721). f, Reduced scratching bouts induced by intradermal pruritogen injection (compound 48/80 (a polymer produced by the condensation of N-methyl-p-methoxyphenethylamine with formaldehyde) test, control, n = 15 mice; TAC1LBX1-Abl, n = 14 mice; two-sided Mann–Whitney rank-sum test, P = 0.002; chloroquine test, control, n = 14 mice; TAC1LBX1-Abl, n = 14 mice; two-sided Mann–Whitney rank-sum test, P = 0.005). Ctrl, control littermates. NS, P > 0.05. Data shown as mean ± s.e.m. (b–d) or mean ± quartile (e, f). Scale bars, 50 μm (a, b), 100 μm (c, d). Source Data
Extended Data Fig. 8 Loss of pinch-induced CPA in female mice in which TAC1LBX1 neurons were ablated.
a, A pinched mouse hindpaw. The alligator clip was applied to the ventral skin surface between the footpad and the heel. b, The experimental programme for pinch-evoked CPA test (for details, see Methods). c, Hindpaw skin pinch, but not sham handling (grabbing without pinching, data not shown), induced CPA in wild-type females. CPA is measured by the change in the amount of time mice stay in the paired chamber before (baseline, t1) and after (test, t2) pinch-evoked conditioning. In three independent batches of wild-type control littermates, a two-sided t-test showed that the second and third batches—but not the first batch—displayed significant reduction of t2 in comparison with t1 (two-sided t-test, batch 1, n = 7 mice, P = 0.0979; batch 2, n = 8 mice, P = 0.0097; batch 3, n = 7 mice, P = 0.0002). Two-way ANOVA analyses of these three batches indicated that pinch-evoked avoidance of the paired chamber (F(1,19) = 36.514, P < 0.001), without showing batch effects (F(2,19) = 0.547, P = 0.587) and interactions (F(2,19) = 0.885, P = 0.429). This suggests that pinching can induce CPA in wild-type females. d, Female mice in which TAC1LBX1 neurons were ablated showed a loss of pinch-induced CPA (control littermates and TAC1LBX1-Abl mice, n = 8 for experiment 1, t-test, *P = 0.031; n = 7 for experiment 2, t-test, **P = 0.001); this was also true for male mice in which TAC1LBX1 neurons were ablated (Fig. 3). Source Data
Extended Data Fig. 9 Optogenetic activation of terminals derived from TAC1Cre-marked neurons around PBN or the medial thalamus.
a–c, Viral infection in lumbar spinal cord TAC1Cre+ neurons and subsequent optogenetic activation of the central terminals in PBN. a, The AAV-DIO-ChR2 virus, which drives the expression of the fusion ChR2–EYFP protein in a Cre-dependent manner, was injected into the lumbar dorsal horn of Tac1cre mouse, and the ascending ChR2–EYFP+ terminals around PBN (dashed line) were visualized by a GFP antibody. These mice are referred to as TAC1Cre–ChR2 mice. Right, immunostaining shows expression of the ChR2–EYFP fusion protein in the lumbar spinal cord. b, Representative images showing double-colour immunostaining, which reveals the ascending projections to the PBN at bregma −5.02 mm. TAC1Cre–ChR2–EYFP+ terminals (dark blue) were co-stained with CGRP (brown). Note that the TAC1Cre–ChR2–EYFP+ fibres pass through a region (b1, arrow) lateral to CGRP+ PBel (b1, brown), and terminated densely in PBsl (b2). c, Representative images showing Fos expression induced by blue-light stimulation, in PBsl of TAC1Cre–ChR2 mice; there are far fewer Fos+ neurons after blue-light stimulation in control TAC1Cre–RFP mice, in which viral injection drove the expression of RFP but not ChR2 (ChR2 mice, n = 4; RFP-control mice, n = 5; two-sided t-test, P = 0.012). Dashed lines indicate the location of the implanted optic fibre in the region above the PBN. d–f, Optogenetic experiments for spinal TAC1 neurons projected to medial thalamic nuclei. d, e, Generation of the intersectional Tac1Cdx2-CatCh+ mice is described in Extended Data Fig. 2b. The optic fibre was implanted above the medial thalamic complex (left). Neuronal activation by blue-light stimulation is indicated by the increase of Fos+ cells in Tac1Cdx2-CatCh mice in comparison with Tac1Cdx2-GFP mice, as shown by representative images and quantitative analyses (Tac1Cdx2-CatCh, n = 3 mice; Tac1Cdx2-GFP, n = 3 mice; two-sided t-test, P = 0.011). f, The Tac1Cdx2-CatCh mice showed a progressive avoidance of the blue-light-paired chamber during two fifteen-minute training trials conducted on two consecutive days (see Methods for details). A two-way ANOVA plus post hoc Bonferroni’s t-test showed a progressive avoidance of the paired chamber (Tac1Cdx2-CatCh mice, n = 10; Tac1Cdx2-GFP control mice, n = 11; trial 1, significant interaction, F(2,38) = 5.067, P = 0.011; trial 2, significant genotype effect, F(1,19) = 6.825, P = 0.017, no interaction, F(2,38) = 0.73, P = 0.489). Source Data
Extended Data Fig. 10 Ablation of TRPV1+ central terminals led to impaired responses to noxious heat or skin burn injury, as well as a reduction of pinch-induced Fos expression in TAC1Cre-marked neurons in the dorsal horn.
a, Mice in which TRPV1+ central terminals were ablated showed a marked increase in the withdrawal latency evoked by the 47-°C hot plate stimulation, with cut off time set at 3 min (vehicle injection versus intrathecal capsaicin injection groups, n = 8 mice for each group, two-sided Mann–Whitney rank-sum test, ***P < 0.001). This is stark contrast to subtle and insignificant changes seen in mice in which TAC1LBX1 neurons were ablated (Fig. 2e). b, Loss of licking behaviour evoked by the 50-°C hot plate stimulation (vehicle injection versus intrathecal capsaicin injection groups, n = 9 mice for each group; licking episodes within one minute, two-sided Mann–Whitney rank-sum test, ***P < 0.001). c, Mice in which TRPV1+ central terminals were ablated also displayed a marked reduction in the licking evoked by hindpaw burn injury (n = 8 mice for each group, licking duration within 30 min of skin burn injury, two-sided t-test, P = 0.001). d, Representative immunostaining images and quantitative analyses showing pinch-evoked Fos expression (green) in the dorsal horn of Tac1cre-tdTomato mice, with or without chemical ablation of TRPV1+ central terminals (n = 4 mice for each group). Note that there is a reduction of pinch-induced Fos expression in TAC1Cre-marked tdTomato+ cells after ablation of TRPV1+ central terminals (two-sided t-test, P = 0.044). TRPV1+ nociceptors are necessary for both reflexes20 (a) and licking (b, c) evoked by noxious heat. Earlier studies have shown that the dorsal root ganglia neurons with highest TRPV1 expression (TRPV1highest), which represent about 10% of TRPV1+ nociceptors52, respond to moderately hot stimulation53. Similar to MRGPRD+ nociceptors, these TRPV1highest neurons innervate exclusively the skin epidermis53, and their development is dependent on the same transcription factor, RUNX152,54. We therefore speculate that these TRPV1highest neurons may be involved with the first-line reflexes evoked by noxious heat. This raises the possibility that there are different subsets of TRPV1+ nociceptors that are associated with reflexes versus sustained pain evoked by noxious heat: future experiments are needed to test this hypothesis. Source Data
The ventral surface of the left hindpaw skin was pinched by an alligator clip. Note the repetitive licking behavior in this control mouse.
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Huang, T., Lin, S., Malewicz, N.M. et al. Identifying the pathways required for coping behaviours associated with sustained pain. Nature 565, 86–90 (2019). https://doi.org/10.1038/s41586-018-0793-8
- Sustained Pain
- Lateral Parabrachial Nucleus
- Skin Pinch
- Generalized Labelled Magnitude Scale (GLMS)
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