A craniofacial-specific monosynaptic circuit enables heightened affective pain

A Publisher Correction to this article was published on 16 March 2018

This article has been updated

Abstract

Humans often rank craniofacial pain as more severe than body pain. Evidence suggests that a stimulus of the same intensity induces stronger pain in the face than in the body. However, the underlying neural circuitry for the differential processing of facial versus bodily pain remains unknown. Interestingly, the lateral parabrachial nucleus (PBL), a critical node in the affective pain circuit, is activated more strongly by noxious stimulation of the face than of the hindpaw. Using a novel activity-dependent technology called CANE developed in our laboratory, we identified and selectively labeled noxious-stimulus-activated PBL neurons and performed comprehensive anatomical input–output mapping. Surprisingly, we uncovered a hitherto uncharacterized monosynaptic connection between cranial sensory neurons and the PBL-nociceptive neurons. Optogenetic activation of this monosynaptic craniofacial-to-PBL projection induced robust escape and avoidance behaviors and stress calls, whereas optogenetic silencing specifically reduced facial nociception. The monosynaptic circuit revealed here provides a neural substrate for heightened craniofacial affective pain.

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Fig. 1: Lateral parabrachial nucleus (PBL) is differentially activated by the same noxious stimulus applied to the face versus hindpaw.
Fig. 2: Capturing and mapping the axonal projection targets of PBL-nociceptive neurons.
Fig. 3: Trans-synaptic labeling of presynaptic neurons for PBL-nociceptive neurons reveals the direct TG→PBL pathway.
Fig. 4: Optogenetic activation of TrpV1-Cre+ sensory axons activates PBL-nociceptive neurons and elicits aversive behavior and stress calls in a real-time place escape/avoidance task.
Fig. 5: Optogenetic silencing of TrpV1-Cre+ axon terminals in PBL selectively reduces face allodynia after capsaicin injection.

Change history

  • 16 March 2018

    In the version of this article initially published, ORCID links were missing for authors Erica Rodriguez, Koji Toda and Fan Wang. The error has been corrected in the HTML and PDF versions of the article.

References

  1. 1.

    Waldman, S. D. Atlas of Common Pain Syndromes (Elsevier Health Sciences, Philadelphia, 2011).

  2. 2.

    Zakrzewska, J. M., Wu, J., Mon-Williams, M., Phillips, N. & Pavitt, S. H. Evaluating the impact of trigeminal neuralgia. Pain 158, 1166–1174 (2017).

    Article  PubMed  Google Scholar 

  3. 3.

    Smith, J. G. et al. The psychosocial and affective burden of posttraumatic neuropathy following injuries to the trigeminal nerve. J. Orofac. Pain 27, 293–303 (2013).

    Article  PubMed  Google Scholar 

  4. 4.

    Schmidt, K., Schunke, O., Forkmann, K. & Bingel, U. Enhanced short-term sensitization of facial compared with limb heat pain. J. Pain 16, 781–790 (2015).

    Article  PubMed  Google Scholar 

  5. 5.

    Schmidt, K. et al. The differential effect of trigeminal vs. peripheral pain stimulation on visual processing and memory encoding is influenced by pain-related fear. Neuroimage 134, 386–395 (2016).

    Article  PubMed  CAS  Google Scholar 

  6. 6.

    Moulton, E. A. et al. Capsaicin-induced thermal hyperalgesia and sensitization in the human trigeminal nociceptive pathway: an fMRI study. Neuroimage 35, 1586–1600 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Hunt, S. P. & Mantyh, P. W. The molecular dynamics of pain control. Nat. Rev. Neurosci. 2, 83–91 (2001).

    Article  PubMed  CAS  Google Scholar 

  8. 8.

    Gauriau, C. & Bernard, J.-F. F. Pain pathways and parabrachial circuits in the rat. Exp. Physiol 87, 251–258 (2002).

    Article  PubMed  Google Scholar 

  9. 9.

    Craig, A. D. Distribution of brainstem projections from spinal lamina I neurons in the cat and the monkey. J. Comp. Neurol. 361, 225–248 (1995).

    Article  PubMed  CAS  Google Scholar 

  10. 10.

    Hermanson, O. & Blomqvist, A. Subnuclear localization of FOS-like immunoreactivity in the rat parabrachial nucleus after nociceptive stimulation. J. Comp. Neurol. 368, 45–56 (1996).

    Article  PubMed  CAS  Google Scholar 

  11. 11.

    Hermanson, O. & Blomqvist, A. Subnuclear localization of FOS-like immunoreactivity in the parabrachial nucleus after orofacial nociceptive stimulation of the awake rat. J. Comp. Neurol. 387, 114–123 (1997).

    Article  PubMed  CAS  Google Scholar 

  12. 12.

    Sakurai, K. et al. Capturing and manipulating activated neuronal ensembles with CANE delineates a hypothalamic social-fear circuit. Neuron 92, 739–753 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. 13.

    Slugg, R. M. & Light, A. R. Spinal cord and trigeminal projections to the pontine parabrachial region in the rat as demonstrated with Phaseolus vulgaris leucoagglutinin. J. Comp. Neurol. 339, 49–61 (1994).

    Article  PubMed  CAS  Google Scholar 

  14. 14.

    Cechetto, D. F., Standaert, D. G. & Saper, C. B. Spinal and trigeminal dorsal horn projections to the parabrachial nucleus in the rat. J. Comp. Neurol. 240, 153–160 (1985).

    Article  PubMed  CAS  Google Scholar 

  15. 15.

    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  PubMed  PubMed Central  CAS  Google Scholar 

  16. 16.

    Gaub, S., Fisher, S. E. & Ehret, G. Ultrasonic vocalizations of adult male Foxp2-mutant mice: behavioral contexts of arousal and emotion. Genes Brain Behav 15, 243–259 (2016).

    Article  PubMed  CAS  Google Scholar 

  17. 17.

    Geerling, J. C. et al. FoxP2 expression defines dorsolateral pontine neurons activated by sodium deprivation. Brain Res. 1375, 19–27 (2011).

    Article  PubMed  CAS  Google Scholar 

  18. 18.

    Ding, Y. Q., Takada, M., Shigemoto, R. & Mizuno, N. Trigeminoparabrachial projection neurons showing substance P receptor-like immunoreactivity in the rat. Neurosci. Res. 23, 415–418 (1995).

    Article  PubMed  CAS  Google Scholar 

  19. 19.

    Tokita, K., Inoue, T. & Boughter, J. D. Jr. Afferent connections of the parabrachial nucleus in C57BL/6J mice. Neuroscience 161, 475–488 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. 20.

    Nishijo, H. & Norgren, R. Parabrachial neural coding of taste stimuli in awake rats. J. Neurophysiol. 78, 2254–2268 (1997).

    Article  PubMed  CAS  Google Scholar 

  21. 21.

    Nakamura, K. & Morrison, S. F. A thermosensory pathway that controls body temperature. Nat. Neurosci. 11, 62–71 (2008).

    Article  PubMed  CAS  Google Scholar 

  22. 22.

    Alhadeff, A. L., Golub, D., Hayes, M. R. & Grill, H. J. Peptide YY signaling in the lateral parabrachial nucleus increases food intake through the Y1 receptor. Am. J. Physiol. Endocrinol. Metab. 309, E759–E766 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. 23.

    Davern, P. J. A role for the lateral parabrachial nucleus in cardiovascular function and fluid homeostasis. Front. Physiol 5, 436 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Menani, J. V., De Luca, L. A. Jr. & Johnson, A. K. Role of the lateral parabrachial nucleus in the control of sodium appetite. Am. J. Physiol. Regul. Integr. Comp. Physiol. 306, R201–R210 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. 25.

    Bester, H., Menendez, L., Besson, J. M. & Bernard, J. F. Spino (trigemino) parabrachiohypothalamic pathway: electrophysiological evidence for an involvement in pain processes. J. Neurophysiol. 73, 568–585 (1995).

    Article  PubMed  CAS  Google Scholar 

  26. 26.

    Bernard, J. F. & Besson, J. M. The spino(trigemino)pontoamygdaloid pathway: electrophysiological evidence for an involvement in pain processes. J. Neurophysiol. 63, 473–490 (1990).

    Article  PubMed  CAS  Google Scholar 

  27. 27.

    Oh, S. W. et al. A mesoscale connectome of the mouse brain. Nature 508, 207–214 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. 28.

    Kohara, K. et al. Cell type–specific genetic and optogenetic tools reveal hippocampal CA2 circuits. Nat. Neurosci. 17, 269–279 (2014).

    Article  PubMed  CAS  Google Scholar 

  29. 29.

    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  PubMed  PubMed Central  CAS  Google Scholar 

  30. 30.

    Panneton, W. M. & Gan, Q. Direct reticular projections of trigeminal sensory fibers immunoreactive to CGRP: potential monosynaptic somatoautonomic projections. Front. Neurosci. 8, 136 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Panneton, W. M., Gan, Q. & Juric, R. Brainstem projections from recipient zones of the anterior ethmoidal nerve in the medullary dorsal horn. Neuroscience 141, 889–906 (2006).

    Article  PubMed  CAS  Google Scholar 

  32. 32.

    Cavanaugh, D. J. et al. Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells. J. Neurosci. 31, 5067–5077 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. 33.

    Mishra, S. K., Tisel, S. M., Orestes, P., Bhangoo, S. K. & Hoon, M. A. TRPV1-lineage neurons are required for thermal sensation. EMBO J 30, 582–593 (2011).

    Article  PubMed  CAS  Google Scholar 

  34. 34.

    Foust, K. D., Poirier, A., Pacak, C. A., Mandel, R. J. & Flotte, T. R. Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons. Hum. Gene Ther. 19, 61–70 (2008).

    Article  PubMed  CAS  Google Scholar 

  35. 35.

    Machida, A. et al. Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice. Mol. Pain 9, 36 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. 36.

    Stanek, E. IV, Rodriguez, E., Zhao, S., Han, B.-X. X. & Wang, F. Supratrigeminal bilaterally projecting neurons maintain basal tone and enable bilateral phasic activation of jaw-closing muscles. J. Neurosci. 36, 7663–7675 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. 37.

    Bellavance, M. A. et al. Parallel inhibitory and excitatory trigemino-facial feedback circuitry for reflexive vibrissa movement. Neuron 95, 673–682.e4 (2017).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  38. 38.

    Madisen, L. et al. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85, 942–958 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. 39.

    Baastrup, C., Jensen, T. S. & Finnerup, N. B. Pregabalin attenuates place escape/avoidance behavior in a rat model of spinal cord injury. Brain Res. 1370, 129–135 (2011).

    Article  PubMed  CAS  Google Scholar 

  40. 40.

    LaBuda, C. J. & Fuchs, P. N. A behavioral test paradigm to measure the aversive quality of inflammatory and neuropathic pain in rats. Exp. Neurol. 163, 490–494 (2000).

    Article  PubMed  CAS  Google Scholar 

  41. 41.

    Zhang, Z. et al. Role of prelimbic GABAergic circuits in sensory and emotional aspects of neuropathic pain. Cell Rep. 12, 752–759 (2015).

    Article  PubMed  CAS  Google Scholar 

  42. 42.

    Daou, I. et al. Optogenetic silencing of Nav1.8-positive afferents alleviates inflammatory and neuropathic pain. eNeuro 3, 0140-15.2016 (2016).

    Article  Google Scholar 

  43. 43.

    Li, B. et al. A novel analgesic approach to optogenetically and specifically inhibit pain transmission using TRPV1 promoter. Brain Res 1609, 12–20 (2015).

    Article  PubMed  CAS  Google Scholar 

  44. 44.

    Chow, B. Y. et al. High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature 463, 98–102 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. 45.

    Sato, M. et al. The lateral parabrachial nucleus is actively involved in the acquisition of fear memory in mice. Mol. Brain 8, 22 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Moffie, D. Late results of bulbar trigeminal tractotomy. Some remarks on recovery of sensibility. J. Neurol. Neurosurg. Psychiatry 34, 270–274 (1971).

    Article  PubMed  CAS  Google Scholar 

  47. 47.

    Rahimpour, S. & Lad, S. P. Surgical options for atypical facial pain syndromes. Neurosurg. Clin. N. Am. 27, 365–370 (2016).

    Article  PubMed  Google Scholar 

  48. 48.

    Romaniello, A., Iannetti, G. D., Truini, A. & Cruccu, G. Trigeminal responses to laser stimuli. Clin. Neurophysiol. 33, 315–324 (2003).

    Article  CAS  Google Scholar 

  49. 49.

    DeSouza, D. D., Moayedi, M., Chen, D. Q., Davis, K. D. & Hodaie, M. sensorimotor and pain modulation brain abnormalities in trigeminal neuralgia: a paroxysmal, sensory-triggered neuropathic pain. PLoS One 8, e66340 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. 50.

    Kuner, R. Central mechanisms of pathological pain. Nat. Med. 16, 1258–1266 (2010).

    Article  PubMed  CAS  Google Scholar 

  51. 51.

    Zhang, F. et al. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat. Protoc. 5, 439–456 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. 52.

    Röhn, T. A. et al. A virus-like particle-based anti-nerve growth factor vaccine reduces inflammatory hyperalgesia: potential long-term therapy for chronic pain. J. Immunol. 186, 1769–1780 (2011).

    Article  PubMed  CAS  Google Scholar 

  53. 53.

    Xu, Z. Z. et al. Inhibition of mechanical allodynia in neuropathic pain by TLR5-mediated A-fiber blockade. Nat. Med. 21, 1326–1331 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. 54.

    Koh, W. U. et al. Perineural pretreatment of bee venom attenuated the development of allodynia in the spinal nerve ligation injured neuropathic pain model; an experimental study. BMC Complement. Altern. Med. 14, 431 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Jennings, J. H. et al. Distinct extended amygdala circuits for divergent motivational states. Nature 496, 224–228 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. 56.

    Stamatakis, A. M. & Stuber, G. D. Activation of lateral habenula inputs to the ventral midbrain promotes behavioral avoidance. Nat. Neurosci. 15, 1105–1107 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. 57.

    Lopes, G. et al. Bonsai: an event-based framework for processing and controlling data streams. Front. Neuroinform 9, 7 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Silva, J. R. et al. Neuroimmune-glia interactions in the sensory ganglia account for the development of acute herpetic neuralgia. J. Neurosci. 37, 6408–6422 (2017).

    Article  PubMed  CAS  Google Scholar 

  59. 59.

    Peng, C. et al. miR-183 cluster scales mechanical pain sensitivity by regulating basal and neuropathic pain genes. Science 356, 1168–1171 (2017).

    Article  PubMed  CAS  Google Scholar 

  60. 60.

    Kim, Y. S. et al. Central terminal sensitization of TRPV1 by descending serotonergic facilitation modulates chronic pain. Neuron 81, 873–887 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. 61.

    Aita, M., Byers, M. R., Chavkin, C. & Xu, M. Trigeminal injury causes kappa opioid-dependent allodynic, glial and immune cell responses in mice. Mol. Pain 6, 8 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. 62.

    Zhang, Y. et al. Identifying local and descending inputs for primary sensory neurons. J. Clin. Invest 125, 3782–3794 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Zhang, Y., Chen, Y., Liedtke, W. & Wang, F. Lack of evidence for ectopic sprouting of genetically labeled Aβ touch afferents in inflammatory and neuropathic trigeminal pain. Mol. Pain 11, 18 (2015).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank J. Takatoh for helping with a method to quantify axon innervation densities, K. Tschida and T. Gibson for helping with vocalization quantification and analysis, and V. Prevosto for helping with statistics. We also thank T. Gibson, M. Fu, K. Tschida, T. Stanek, V. Prevosto, and R. R. Ji for providing input and support throughout the project, and S. Lisberger and R. Mooney for critical reading of this manuscript. E.R. is supported by a F31 DE025197-03 fellowship. Y.C. is supported by K12DE022793. W.L. is supported by DE018549. This work is supported by NIH Grant DP1MH103908 to F.W.

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Contributions

F.W. and E.R. conceived the idea and designed the experiments. E.R. performed the majority of the experiments and data analysis. K.S. performed some independent CANE capture experiments, bilateral fiber implantations and the place escape/avoidance (PEA) behavioral experiments. K.T. analyzed PEA results (blind to genotype). J.X. performed immunohistochemistry, quantified axon projections, and quantified cells in Fos and trans-synaptic experiments (blind to experimental conditions). Y.C. performed all the face and hindpaw von Frey assays (blind to genotypes). D.R. quantified cells in a subset of colocalization experiments. S.Z. produced all the CANE-LV and CANE-RV viruses. B.-X.H. took care of mouse husbandry and genotyping. H.Y. and W.L. provided critical equipment and reagents. F.W. and E.R. wrote the manuscript with help from W.L.

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Correspondence to Fan Wang.

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Integrated supplementary information

Supplementary Figure 1 Fos expression patterns in the PBL after different types of noxious injections into the right whisker pad (related to Fig. 1)

(a) Anti-Fos staining was performed 90 minutes after each injection. Blue; DAPI stain. Scale bar, 100 µm. (b) Quantification of Fos+ neurons in the PBL. (n = 3,4,4,4; one-way ANOVA; Home Cage vs. Saline: *P = 0.0437; Home Cage vs. Capsaicin: **P = 0.0014; Home Cage vs. Formalin: ***P = 0.0001; F3, 11 = 18.25) Data are mean ± SEM. (c) Anti-Fos staining was performed on Sp5C 90 minutes after formalin injection. Blue; DAPI stain. Scale bar, 100 µm

Supplementary Figure 2 Molecular characterization of PBL-nociceptive neurons (related to Fig. 1)

(a) Two-color fluorescent in situ hybridization showing formalin-activated (Fos+, green) and Vglut2-expressing neurons (right) and Gad1/2-expressing neurons (left) (both magenta) in the PBL. Scale bar, 20 µm. (b) Quantification of co-expression of Fos+ neurons and Vglut2 and Gad1/2-expressing neurons in the PBL. (n = 3; two-tailed paired Student’s t test; **P = 0.001; t2= 29.73). Data are mean ± SEM. (c) Left panel, staining of formalin-activated neurons (anti-Fos, green) and CGRP+ neurons (magenta). Right panel, staining of formalin activated neurons (anti-Fos, green) and FoxP2+ neurons (magenta). Scale bar, 100 µm. (d) Quantification of co-expression of Fos+ neurons and FoxP2+ and CGRP+ neurons in PBL-dorsal, PBL-ventral, and total PBL. (n = 4; two-tailed paired Student’s t test; P = 0.0544, *P = 0.0496, P = 0.1503; t2 = 4.109, t2 = 4.32, t2 = 2.279). Data are mean ± SEM 

Supplementary Figure 3 Additional evidence for the specificity of CANE captured PBL-nociceptive neurons (related to Fig. 2)

. Representative image of CANE captured PBL-nociceptive neurons (Green) and generally labeled mCherry+ PBL neurons (magenta) after co-injection of CANE-LV-Cre; AAV-flex-GFP; AAV-tdTomato. (n = 4 hemispheres in 2 mice). Scale bar, 50 µm (both low and high mag)

Supplementary Figure 4 Labeling of TrpV1-Cre+ primary sensory neurons but not CNS neurons (related to Fig. 3)

(a) Schematic illustration and timeline of intraperitoneal injection in 1-2 day old TrpV1-Cre pup with AAV-CAG-flex-GFP. Four weeks after injection, TrpV1Cre::GFP mouse was injected with capsaicin in the whisker pad and stained for Fos (n = 3 mice). (b-d) Representative images of a cortical section (b), Sp5C (c), and DRG (d) from a TrpV1-Cre mouse intraperitoneally injected with AAV-CAG-flex-GFP and stained for Fos (magenta). Note that there is no GFP expressing neuronal cell bodies in CNS. Scale bar, (b, c) 500 µm and (d) 50 µm

Supplementary Figure 5 Selective labeling of PBL projecting TrpV1-Cre+ neurons using a retrograde-FlpO and TrpV1-Cre intersectional strategy revealed that these neurons project to both PBL and Sp5C

(a) Schematic illustration of the intersectional strategy (retrograde FlpO together with TrpV1-Cre) to selectively label TrpV1-Cre+ neurons projecting to PBL. (b-d) Representative images of sparse labeling results using RG-LV-hSyn-DIO-FlpO in combination with TrpV1-Cre in Ai65 reporter (n=6): (b), labeled axon terminals in PBL. (c), few labeled TG neuron cell bodies. (d), labeled axon terminals in Sp5C. Scale bars, 50, 20, 50μm. (e-h) Representative images of dense labeling results using RG-LV-hSyn-FlpO in combination with TrpV1-Cre in Ai65 reporter (n = 4): (e), labeled axon terminals in PBL. (f), labeled TG neurons. (g), labeled axon terminals in Sp5C. (h), labeled peripheral axon terminals in lower lip and whisker pad. Scale bars, 50μm (e), 50μm (f), 100μm (g), 50μm (h)

Supplementary Figure 6 EPSC characterization of PBL neurons receiving direct TrpV1Cre::ChR2+ TG afferent inputs (related to Fig. 4)

. (a-c) Quantification of the rise time, half-width, and decay time of the photo-stimulating TrpV1Cre::ChR2 axons evoked EPSCs in recorded PBL neurons (n = 15 cells). Data are mean ± SEM. (d) Correlation of the TrpV1Cre::ChR2 evoked EPSC amplitude over the onset latency of the EPSC. (Nonlinear regression; n = 15 cells)

Supplementary Figure 7 Optogenetic activation of ChR2-expressing TG afferents in the PBL induces place avoidance in a conditioned place aversion (CPA) assay

(a) Schematic illustration of the conventional conditioned place aversion (CPA) test. (b) Representative spatial tracking map showing the location of an experimental mouse before and after optogenetic stimulation of TrpV1Cre::ChR2+ axon terminals in the PBL in the preferred chamber. (c) Quantification of time the experimental group spent in preferred chamber before and after optogenetic stimulation (n = 7 two-tailed paired Student’s t test; **P = 0.0080; t6 =3.899). Data are mean ± SEM. (d) Representative spatial tracking map showing the location of an experimental mouse before and after light illumination of TrpV1Cre::GFP+ axon terminals in the PBL in the preferred chamber. (e) Quantification of time the control group spent in preferred chamber before and after light illumination (n = 6, two-tailed paired Student’s t test; P = 0.2576; t4 =1.319). Data are mean ± SEM

Supplementary Figure 8 Optogenetic activation of ChR2-expressing TG afferents in the PBL induces vocalization (related to Fig. 4).

Representative spectrograms of induced audible vocalizations of an adult mouse during photo activation (20ms pulses at 10Hz) of TrpV1Cre::ChR2+ axon terminals within the PBL. Vocalization stops when laser light turns off (n = 8 mice)

Supplementary Figure 9 Post-hoc analysis after optogenetic stimulation of TrpV1Cre::ChR2+ axons in PBL (related to Fig. 4)

(a) Representative image from a TrpV1-Cre mouse (n = 5 mice) intraperitoneally injected with AAV-flex-ChR2-EYFP which underwent photo stimulation. Numerous Fos+ (magenta) neurons in PBL were observed after photo stimulation of TrpV1Cre::ChR2+ axon terminals (green). Scale bar, 100 µm. (b) Relatively few Sp5C neurons expressed Fos (magenta) after photo stimulation of TrpV1Cre::ChR2+ axon terminals in the PBL. Scale bar, 100 µm. (c) Representative image from a TrpV1-Cre mouse intraperitoneally injected with AAV-flex-GFP which underwent photo illumination (n = 3 mice). Few PBL neurons expressed Fos (magenta, background expression) after photo stimulation of TrpV1Cre::GFP+ axon terminals (green). Scale bar, 100 µm

Supplementary Figure 10 Schematic illustration of trigeminal sensory pathways

Schematic summary for output targets of tactile (green) and nociceptive (red) trigeminal ganglion (TG) sensory neurons, including the newly discovered TG\(\to \)PBL projection. Schematic illustration demonstrates location of where DREZ (dorsal root entry zone coagulation) is performed to lesion the TG\(\to \)Sp5C pathway to treat refractory craniofacial pain. Note that DREZ lesion will not affect the TG\(\to \)PBL projection. Pr5, principal sensory trigeminal nucleus; Sp5O; trigeminal nucleus, oral; Sp5I, trigeminal nucleus, interpolaris; Sp5C, trigeminal nucleus, caudalis; DREZ, dorsal root entry zone coagulation

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Supplementary Figures 1–10

Life Sciences Reporting Summary

Supplementary Video 1

Optogenetic activation of TrpV1Cre::ChR2+ TG afferents in the PBL in a real-time place escape/avoidance test (related to Fig. 4). Photo activation of TrpV1Cre::ChR2+ TG axon terminals within the PBL elicits escaping from the stimulation chamber to the opposite chamber to stop the stimulation. After mouse escapes to the non-stimulated chamber, it moves less and spends more time in the chamber.

Supplementary Video 2

Photo illumination of TrpV1Cre::GFP+ TG afferents in the PBL in a real-time place escape/avoidance test (related to Fig. 4). Photo illumination of TrpV1Cre::GFP+ TG axon terminals within the PBL has no observable behavioral effects.

Supplementary Video 3

Optogenetic activation of TrpV1Cre::ChR2+ TG afferents in the PBL in a circular chamber to record vocalization (related to Fig. 4). Photo activation of TrpV1Cre::ChR2+ TG axon terminals within the PBL induces audible distress vocalization. Vocalization stops when laser light turns off.

Supplementary Video 4

Photo illumination of TrpV1Cre::GFP+ TG afferents in the PBL in a circular chamber to record vocalization (related to Fig. 4). Photo illumination of TrpV1Cre::GFP+ TG axon terminals within the PBL does not induce any vocalizations.

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Rodriguez, E., Sakurai, K., Xu, J. et al. A craniofacial-specific monosynaptic circuit enables heightened affective pain. Nat Neurosci 20, 1734–1743 (2017). https://doi.org/10.1038/s41593-017-0012-1

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