A craniofacial-specific monosynaptic circuit enables heightened affective pain

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 (PB_L), 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 PB_L neurons and performed comprehensive anatomical input-output mapping. Surprisingly, we uncovered a hitherto uncharacterized monosynaptic connection between cranial sensory neurons and the PB_L-nociceptive neurons. Optogenetic activation of this monosynaptic craniofacial-to-PB_L 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.

Figure 1. Lateral parabrachial nucleus (PB_L) is differentially activated by the same noxious stimulus applied to the face versus hindpaw

(a) Schematic illustration of Fos induction protocol. Ninety minutes after 10 µL 4% formalin was injected, brainstem slices containing PB_L were stained for Fos expression. (TG, trigeminal ganglion; Sp5C, trigeminal nucleus, caudalis; DRG, dorsal root ganglion; S.C., spinal cord) (b) Representative images of Fos⁺ neurons in PB_L after formalin injection into right whisker pad (top) and right hindpaw (bottom). Large white dash circle (left) indicates the entire structure of PB_L, whereas small white dash circle (right) indicates ventral region of PB_L including PB-el. Blue; DAPI stain. Scale bar, 200 µm. (c) Total numbers of Fos⁺ neurons in PB_L on both sides combined (n = 4, 3; two-tailed unpaired student’s t test; *P = 0.0445; t_4.962 =2.674). (d) Numbers of Fos⁺ neurons in ipsilateral (magenta) and contralateral (teal) PB-el in mice unilaterally injected with formalin into one whisker pad (n = 4) or one hindpaw (n = 3 mice; two-way ANOVA; W: P = 0.0533; H: **P = 0.0090; F_{1, 5} = 32.75). Data are mean ± SEM.

Figure 2. Capturing and mapping the axonal projection targets of PB_L-nociceptive neurons

(a) Schematic illustration of strategy to express GFP in nociceptive relay PB_L neurons in Fos^TVA mice using CANE. (b-g) Examination of CANE-captured neurons activated by the first stimulus (magenta) versus Fos⁺ neurons activated by the second stimulus (green) in the PB_L. In all six paradigms, CANE method was used to capture neurons activated by stimulus/no stimulus, and 2 weeks later, Fos was induced by the second stimulus. Blue, DAPI. Scale bars, 10 µm. (h-j) The percentage of Fos⁺ neurons among CANE⁺ neurons. Data are mean ± SEM. (from left to right: ((h) n =4, 9, 7, 4; one-way ANOVA; ****P = <0.0001, **P = 0.0005, P = 0.3952, P = 0.3223; **P = 0.0005, *P = 0.0047; F_{3, 20} = 12.49 (i) n = 5, 5, 9; one-way ANOVA; ****P = <0.0001, ****P = <0.0001, P = 0.6876; F_{2, 17} = 52.17 (j) n = 3, 3; two-tailed unpaired student’s t test; P = 0.2759; t_3.505=1.289). (k-p) Representative images of axonal projections from captured formalin activated PB_L (magenta) in several brain nuclei expressing Fos (green) induced by formalin. (Inset: Schematic of coronal view of location [in red box] in brain). * in panel k denotes very large terminal boutons from labeled PB_L axons in BNST, some of boutons surround the Fos⁺ BNST neuron cell bodies. (q) Quantification of normalized density of innervations (total pixels divided by the area of each nucleus; n = 3). All data shown are mean ± SEM. (r) Schematic summary for output targets of PB_L-nociceptive neurons. BNST, bed nucleus of the stria terminalis; PVH, paraventricular hypothalamic nucleus; PVT, paraventricular nucleus of the thalamus; CeA_C, central amygdalar nucleus, capsular part; SN_PC, substantia nigra pars compacta; PAG_vl, ventrolateral periaqueductal gray; NST, nucleus solitary tract; IRt, intermediate reticular tract. Scale bars, (k, o) 20 µm. (l-n, p) 50 µm. (n =3) Data are mean ± SEM.

Figure 3. Transsynaptic labeling of presynaptic neurons for PB_L-nociceptive neurons reveals the direct TG→PB_L pathway

(a) Schematic illustration for transsynaptic tracing of presynaptic inputs to PB_L-nociceptive neurons. (b) Representative image of CANE-RV-mCherry-infected PB_L-nociceptive neurons. Green, PB_L-nociceptive neurons expressing TVA and RG. Red, RV-mCherry⁺. Yellow, starter cells. Scale bar, 10 µm. (c-l) Representative images of transsynaptically labeled neurons in several brain regions. Scale bars, (c-h, k-l) 50 µm (i) 100 µm (j) 20 µm (m) Quantification of transsynaptically labeled neurons in each brain area contralateral (teal) and ipsilateral (magenta) to injected site after whisker pad formalin injection and (n) after hindpaw formalin injection. The value is normalized against the number of starter neurons and averaged across animals. Data are mean ± SEM (n = 6; n = 3). (o) Schematic summary for input sources for PB_L-nociceptive neurons. BNST, bed nucleus of the stria terminalis; PVH, paraventricular of the hypothalamus; LHA, lateral hypothalamus; CeA, central amygdalar nucleus, medial; SN_PC, substantia nigra pars compacta; PAG, periaqueductal gray; DRN, dorsal raphe nucleus; Reticular Nuclei: (PRn, pontine reticular nuclei; IRt, intermediate reticular tract; PCRt, parvicellular reticular tract, MRn, medullary reticular nuclei; GRn, gigantocellular reticular nuclei); NST, nucleus solitary tract; Sp5C, trigeminal nucleus, caudalis; TG, trigeminal ganglion; DRG, dorsal root ganglion. S.C., spinal cord (dorsal horn). (p) Molecular characterization of transsynaptically labeled trigeminal ganglion (TG) neurons. Green; bottom to top: IB4⁺, CGRP⁺, NF200⁺, TrpV1⁺. Left, colocalized transsynaptically labeled TG neurons. Right, non-colocalized labeled TG neurons. Scale bar, 20 µm. (q) Percentage of transsynaptically labeled trigeminal ganglion neurons expressing IB4, CGRP, NF200, or TrpV1. (n = 8; one-way ANOVA; *P = 0.0135, ***P = 0.0008, *P = 0.0468, ****P = <0.0001, ****P = <0.0001, P = 0.4653; F_{3, 27} = 22.7). Data are mean ± SEM. (r) Schematic illustration and timeline of intraperitoneal injection in 1–2 day old TrpV1-Cre pup with AAV9-CAG-flex-GFP. Four weeks after injection, TrpV1Cre::GFP mouse was injected with capsaicin in the whisker pad and stained for Fos (n = 4 mice). (s) Representative image of trigeminal ganglion with TrpV1Cre::GFP⁺ neurons. Scale bar, 200 µm. (t) Representative image of PB_L with TrpV1Cre::GFP⁺ axon terminals (Green) and capsaicin-induced Fos⁺ neurons (magenta). Scale bar, 50 µm (high mag).

Figure 4. Optogenetic activation of TrpV1-Cre⁺ sensory axons activates PB_L-nociceptive neurons and elicits aversive behavior and stress calls in a real-time place escape/avoidance task

(a) Schematic illustration of intraperitoneal injection of a 1–2 day old TrpV1-Cre pup (n = 3), followed by optogenetic-assisted whole cell patch-clamp recording from a PB_L neuron in acute brain slices (b) Representative traces from a cell showing no light-evoked IPSC at a holding potential of 10mV, but observed to have light-evoked EPSC at a holding of −65mV. Cell, held at −65 mV, was bath applied with 1µM TTX, followed by 100 µM 4-AP and 1µM TTX, showing a light-evoked monosynaptic EPSC. (c) Averaged current amplitude is shown. Data are mean ± SEM. (closed circles, individual cells, n = 15). (d) Representative high-mag image of TrpV1Cre::ChR2⁺ axon terminals and CANE-RV-mCherry captured PB_L-pain neurons. (n = 3 mice; Scale bar, 50 µm). (e) Representative of an mCherry⁺ PB_L-pain neuron recorded to have light-evoked EPSC at a holding of −65mV. Cell was bath applied with 1µM TTX, followed by 100 µM 4-Ap and 1µM TTX, showing a light-evoked monosynaptic EPSC. (f) Averaged current amplitude is shown. Data are mean ± SEM. (closed circles, individual cells, n = 6). (g) Schematic illustration of real-time place escape/avoidance (PEA) test. (h) Representative spatial tracking map showing the location of an experimental mouse before, during, and after optogenetic stimulation of TrpV1Cre::ChR2⁺ axon terminals in the PB_L in the preferred chamber. (i) Representative spatial tracking map showing the location of a control mouse before, during, and after illuminating TrpV1Cre::GFP⁺ axon terminals in the PB_L in the preferred chamber. (j) Percentage of preference (per 30 seconds) the experimental and control groups had before, during, and after optogenetic stimulation (n = 8 & 3) shown across time (min). Data are mean ± SEM. (k) Quantification of time theTrpV1Cre::ChR2 group spent in preferred chamber before, during, and after optogenetic stimulation (n = 8 one-way repeated measures ANOVA; ****P = <0.0001, *P = 0.0128, ****P = <0.0001; F_{2, 14} = 49.41). Data are mean ± SEM. (l) Quantification of time the TrpV1Cre::GFP group spent in preferred chamber before, during, and after light illumination (n = 3; one-way repeated measures ANOVA; P = 0.8867, P = 0.6377, P = 0.8886; F_{2, 6} = 0.4412). Data are mean ± SEM. (m) Schematic illustration of vocalization recording chamber. (n) Quantification of frequency of pips induced by optogenetic stimulation of TrpV1Cre::ChR2 (experimental) or TrpV1Cre::GFP (control) axon terminals in the PB_L. Data are mean ± SEM. (ChR2, n = 8; GFP, n = 3; two-tailed unpaired student’s t test; **P = <0.0001; t₇ =10.13).

Figure 5. Optogenetic silencing of TrpV1-Cre⁺ axon terminals in PB_L selectively reduces face allodynia after capsaicin injection

(a) Schematic illustration of intraperitoneal injection of a 1–2 day old TrpV1-Cre pup followed by a face and hindpaw von Frey tests in the same individual mice in TrpV1Cre::eArch (n = 9) and TrpV1Cre::GFP groups (n = 8). The order of face versus hindpaw tests was randomized. Each mouse was tested before and after 10 µL 4% capsaicin was injected into either face or hindpaw. (b) Representative post-hoc image of TrpV1Cre::eArch⁺ axon terminals in PB_L and labeled TrpV1Cre::eArch⁺ cell bodies in TG (n = 9 mice; Scale bars, 50 µm). (c) Quantification of mechanical thresholds of face withdrawal responses in von Frey tests. Measurements were taken before and after capsaicin injection into right whisker pad, as well as without and with optogenetic silencing in TrpV1Cre::eArch (n = 9) or in control TrpV1Cre::GFP groups (n = 8, two-way repeated measures ANOVA; (eArch vs. GFP) P = >0.9999, P = >0.9999, P = >0.9999, *P = 0.0440; (no light vs light) eArch: P = >0.9999, **P = 0.0046, GFP: P = >0.9999, P = >0.9999; F (3, 45) = 2.671). Data are mean ± SEM. (d) Quantification of mechanical thresholds of hindpaw withdrawal responses in von Frey tests. Measurements were taken before and after capsaicin injection into right hindpaw, as well as without and with optogenetic silencing in TrpV1Cre::eArch (n = 9) or in TrpV1Cre::GFP groups (n = 8, two-way repeated measures ANOVA; (Arch vs. GFP) P = >0.9999, P = >0.9999, P = >0.9999, >0.9999; (no light vs light) Arch: P = >0.9999, P=>0.9999, GFP: P = >0.9999, P = >0.9999; F (3, 45) = 0.03048). Data are mean ± SEM. (e) Schematic illustration of real-time place preference (RTPP) test of mouse injected with capsaicin into left whisker pad. (f) Quantification of time the experimental group spent in non-preferred chamber before capsaicin, after capsaicin, and without or with optogenetic silencing (n = 6 one-way repeated measures ANOVA; P = 0.5356, *P = 0.0174, **P = 0.0031; F_{2, 10} = 10.92). Data are mean ± SEM. (g) Quantification of time the control group spent in non-preferred chamber before capsaicin, after capsaicin, and without or with optogenetic silencing (n = 7; one-way repeated measures ANOVA; P = 0.7320, P = 0.2086, P = 0.5537; F_{2, 10} = 1.695). Data are mean ± SEM.