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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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.
The authors declare no competing financial interests.
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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
(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
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
Supplementary Figures 1–10
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.
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.
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.
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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