Identifying the pathways required for coping behaviours associated with sustained pain (original) (raw)

Data availability

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/).

References

1. 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).
   Article Google Scholar
2. Young, R. F. et al. Gamma knife thalamotomy for the treatment of persistent pain. Stereotact. Funct. Neurosurg. 64, 172–181 (1995).
   Article Google Scholar
3. Price, D. D. Central neural mechanisms that interrelate sensory and affective dimensions of pain. Mol. Interv. 2, 392–403, 339 (2002).
   Article Google Scholar
4. 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).
   Article CAS Google Scholar
5. Todd, A. J. Neuronal circuitry for pain processing in the dorsal horn. Nat. Rev. Neurosci. 11, 823–836 (2010).
   Article CAS Google Scholar
6. Bourane, S. et al. Identification of a spinal circuit for light touch and fine motor control. Cell 160, 503–515 (2015).
   Article CAS Google Scholar
7. Head, H. & Holmes, H. J. Sensory disturbances from cerebral lesions. Brain 34, 102–154 (1911).
   Article Google Scholar
8. 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).
   Article CAS Google Scholar
9. Rodriguez, E. et al. A cranio-facial-specific monosynaptic circuit enables hieghtened affective pain. Nat. Neurosci. 20, 1734–1743 (2017).
   Article CAS Google Scholar
10. 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).
    Article ADS Google Scholar
11. Mu, D. et al. A central neural circuit for itch sensation. Science 357, 695–699 (2017).
    Article ADS CAS Google Scholar
12. Oh, S. W. et al. A mesoscale connectome of the mouse brain. Nature 508, 207–214 (2014).
    Article ADS CAS Google Scholar
13. Duan, B. et al. Identification of spinal circuits transmitting and gating mechanical pain. Cell 159, 1417–1432 (2014).
    Article CAS Google Scholar
14. Bourane, S. et al. Gate control of mechanical itch by a subpopulation of spinal cord interneurons. Science 350, 550–554 (2015).
    Article ADS CAS Google Scholar
15. Melzack, R. & Wall, P. D. Pain mechanisms: a new theory. Science 150, 971–979 (1965).
    Article ADS CAS Google Scholar
16. Duan, B., Cheng, L. & Ma, Q. Spinal circuits transmitting mechanical pain and itch. Neurosci. Bull. 34, 186–193 (2018).
    Article Google Scholar
17. 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).
    Article CAS Google Scholar
18. Vandewauw, I. et al. A TRP channel trio mediates acute noxious heat sensing. Nature 555, 662–666 (2018).
    Article ADS CAS Google Scholar
19. 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).
    Article CAS Google Scholar
20. 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).
    Article ADS CAS Google Scholar
21. 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).
    Article CAS Google Scholar
22. 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).
    Article CAS Google Scholar
23. Malin, S. et al. TRPV1 and TRPA1 function and modulation are target tissue dependent. J. Neurosci. 31, 10516–10528 (2011).
    Article CAS Google Scholar
24. Cheng, L. et al. Identification of spinal circuits involved in touch-evoked dynamic mechanical pain. Nat. Neurosci. 20, 804–814 (2017).
    Article CAS Google Scholar
25. 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).
    CAS PubMed Google Scholar
26. 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).
    Article CAS Google Scholar
27. 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).
    Article Google Scholar
28. 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).
    Article CAS Google Scholar
29. 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).
    Article Google Scholar
30. 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).
    Article Google Scholar
31. Niederkofler, V. et al. Identification of serotonergic neuronal modules that affect aggressive behavior. Cell Rep. 17, 1934–1949 (2016).
    Article CAS Google Scholar
32. Britz, O. et al. A genetically defined asymmetry underlies the inhibitory control of flexor–extensor locomotor movements. eLife 4, e04718 (2015).
    Article Google Scholar
33. Liu, Y. et al. VGLUT2-dependent glutamate release from nociceptors is required to sense pain and suppress itch. Neuron 68, 543–556 (2010).
    Article CAS Google Scholar
34. 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).
    Article CAS Google Scholar
35. 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).
    Article CAS Google Scholar
36. 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).
    Article ADS CAS Google Scholar
37. Pogorzala, L. A., Mishra, S. K. & Hoon, M. A. The cellular code for mammalian thermosensation. J. Neurosci. 33, 5533–5541 (2013).
    Article CAS Google Scholar
38. Green, B. G. et al. Evaluating the ‘Labeled Magnitude Scale’ for measuring sensations of taste and smell. Chem. Senses 21, 323–334 (1996).
    Article ADS CAS Google Scholar
39. Bartoshuk, L. M. et al. Valid across-group comparisons with labeled scales: the gLMS versus magnitude matching. Physiol. Behav. 82, 109–114 (2004).
    Article CAS Google Scholar
40. 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).
    Article CAS Google Scholar
41. 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).
    Article CAS Google Scholar
42. 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).
    Article CAS Google Scholar
43. Kleinlogel, S. et al. Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh. Nat. Neurosci. 14, 513–518 (2011).
    Article CAS Google Scholar
44. 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).
    Article CAS Google Scholar
45. Story, G. M. et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112, 819–829 (2003).
    Article CAS Google Scholar
46. Bautista, D. M. et al. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 124, 1269–1282 (2006).
    Article CAS Google Scholar
47. 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).
    Article Google Scholar
48. 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).
    Article CAS Google Scholar
49. Doehring, A. et al. Effect sizes in experimental pain produced by gender, genetic variants and sensitization procedures. PLoS ONE 6, e17724 (2011).
    Article ADS CAS Google Scholar
50. 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).
    Article Google Scholar
51. Benabid, A. L. & Jeaugey, L. Cells of the rat lateral habenula respond to high-threshold somatosensory inputs. Neurosci. Lett. 96, 289–294 (1989).
    Article CAS Google Scholar
52. Abdel Samad, O. et al. Characterization of two Runx1-dependent nociceptor differentiation programs necessary for inflammatory versus neuropathic pain. Mol. Pain 6, 45 (2010).
    PubMed Google Scholar
53. Kiasalari, Z. et al. Identification of perineal sensory neurons activated by innocuous heat. J. Comp. Neurol. 518, 137–162 (2010).
    Article CAS Google Scholar
54. Chen, C. L. et al. Runx1 determines nociceptive sensory neuron phenotype and is required for thermal and neuropathic pain. Neuron 49, 365–377 (2006).
    Article CAS Google Scholar

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Acknowledgements

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.

Reviewer information

Nature thanks F. Wang and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

1. These authors contributed equally: Tianwen Huang, Shing-Hong Lin

Authors and Affiliations

1. Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA
   Tianwen Huang, Shing-Hong Lin, Yan Zhang, Ying Zhang & Qiufu Ma
2. Department of Neurobiology, Harvard Medical School, Boston, MA, USA
   Tianwen Huang, Shing-Hong Lin & Qiufu Ma
3. Department of Anesthesiology, Yale University School of Medicine, New Haven, CT, USA
   Nathalie M. Malewicz & Robert H. LaMotte
4. Institute of Acupuncture and Moxibustion, Fudan Institutes of Integrative Medicine; Department of Integrative Medicine and Neurobiology, School of Basic Medical Science, Fudan University, Shanghai, China
   Yan Zhang
5. Cell Electrophysiology Laboratory, Wannan Medical College, Wuhu, China
   Yan Zhang
6. Neuroscience Research Institute and Department of Neurobiology, School of Basic Medical Sciences, Key Laboratory for Neuroscience, Ministry of Education/National Health and Family Planning Commission, Peking University, Beijing, China
   Ying Zhang
7. Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA
   Martyn Goulding

Authors

1. Tianwen Huang
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2. Shing-Hong Lin
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3. Nathalie M. Malewicz
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4. Yan Zhang
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5. Ying Zhang
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6. Martyn Goulding
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7. Robert H. LaMotte
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8. Qiufu Ma
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Contributions

T.H., S.-H.L., N.M.M., Yan Zhang and Ying Zhang performed the experiments and data analyses, Q.M., R.H.L. and M.G supervised the whole study, and T.H., S.-H.L., Q.M., R.H.L. and M.G. wrote the manuscript.

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Correspondence toQiufu Ma.

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Extended data figures and tables

Extended Data Fig. 1 Neurotransmitter phenotypes and central projection by spinal TAC1Cre+ neurons.

a–c, Lumbar spinal sections from P30 Tac1 cre -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 Tac1 cre mice and Cdx2 Flpo 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 Tac1 Cdx2 -tdTomato mice. e, Representative sections through the spinal cord of Tac1 Cdx2 -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 Cdx2 Flpo -tdTomato mice (left, n = 3) and Tac1 Cdx2 -tdTomato mice (right, n = 3). Cdx2 Flpo -tdTomato mice were generated by crossing Cdx2 Flpo mice with Flpo-dependent Rosa26 FSF-tdTomato reporter mice. Among 25-μm-thick sections of VPL from Tac1 Cdx2 -tdTomato mice, only 12% (3 out of 26) showed sparse tdTomato signals, whereas 100% (26 out of 26) of sections from Cdx2 Flpo -tdTomato mice showed robust tdTomato signals (_χ_2 test, _χ_20.95,(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 Cdx2 Flpo -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 Cdx2 Flpo -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 Cdx2 Flpo -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).

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Extended Data Fig. 2 Functional connections of Tac1 _cre_-marked neurons to neurons in PBsl.

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 Tac1 cre 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 Tac1 cre and Cdx2 Flpo, with the resulting triple heterozygous mice referred to as Tac1 _Cdx2_-CatCh mice. Triple heterozygous Tac1 _Cdx2_-GFP mice—generated by crossing Tac1 cre and Cdx2 Flpo 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 Tac1 _Cdx2_-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; _χ_2 test, _χ_20.95,(1) = 4.615, P = 0.032; Tac1 Cdx2 -CatCh mice, n = 2).

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Extended Data Fig. 3 Retrograde labelling of spinal Tac1 _cre_-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 Tac1 cre -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 Tac1 cre -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

Extended Data Fig. 6 Skin-pinch-evoked sustained pain in humans.

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.

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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 Tac1 cre -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).

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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, _t_1) and after (test, _t_2) 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 _t_2 in comparison with _t_1 (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).

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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 Tac1 cre 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 Tac1 Cdx2 -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 Tac1 Cdx2 -CatCh mice in comparison with Tac1 _Cdx2_-GFP mice, as shown by representative images and quantitative analyses (Tac1 Cdx2 -CatCh, n = 3 mice; Tac1 Cdx2 -GFP, n = 3 mice; two-sided _t_-test, P = 0.011). f, The Tac1 Cdx2 -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 (Tac1 Cdx2 -CatCh mice, n = 10; Tac1 Cdx2 -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).

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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 Tac1 cre -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.

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Huang, T., Lin, SH., 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

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• Received: 02 January 2018
• Accepted: 05 November 2018
• Published: 10 December 2018
• Issue Date: 03 January 2019
• DOI: https://doi.org/10.1038/s41586-018-0793-8

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