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

Identifying the pathways required for coping behaviours associated with sustained pain

Tianwen Huang et al. Nature. 2019 Jan.

Abstract

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 nuclei1-3. 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.

PubMed Disclaimer

Conflict of interest statement

Author information. All authors have no conflicting interests. Correspondence and requests for materials should be addressed to Q.M.

Figures

Extended Data Fig. 1

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 labeled by tdTomato expression, 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 hybridization. Right panels represent 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% are VGLUT2+ excitatory neurons, 6.1 ± 1.1% are GAD67+ GABAergic inhibitory neurons, and 4.5 ± 0.9% are GlyT2+ glycinergic inhibitory neurons. data were presented as S.E.M. d, Intersectional genetic strategy for driving tdTomato expression in spinal Tac1Cdx2 neurons defined by co-expression of Tac1Cre and Cdx2Flpo. It had been reported previously that Cdx2Flpo drives reporter expression from the cervical spinal cord all the way to the most caudal spinal cord. By crossing Tac1 _Cr_e mice and Cdx2 Flpo mice with intersectional Ai65 reporter mice, only spinal Tac1Cre+ neurons co-expressed Flpo drove tdTomato expression, referred to as Tac1 Cdx2 -tdTomato mice. e, Representative sections through the spinal cord of Tac1 Cdx2 -tdTomato mice (n = 3), showing tdTomato+ neurons are not detected at the most rostral cervical levels or in the brain (data not shown), but are detected at the lumbar levels. f, Representative coronal sections (25 μm thick, prepared by cryostat, in comparison with Fig. 1c, which was 100 μm thick, 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 VPL sections from Tac1 Cdx2 -tdTomato mice, only 12% (3/26) showed sparse tdTomato signals, while 100% (26/26) of sections from Cdx2 Flpo -tdTomato mice showed robust tdTomato signals (CHI-test, χ2.95,(1) = 41.241, P < 0.001). It should be noted that due to restriction of Cdx2Flpo to the spinal cord, no tdTomato signals were detected in the VPM of Cdx2 Flpo -tdTomato mice, since VPM is innervated by neurons located in the trigeminal nuclei or dorsal column nuclei that were not labeled 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 the whole spinal ascending fibers in the medial thalamic complex. i, Representative coronal sections (25 μm) of parabrachial nuclei (PBN) from P30 Cdx2 Flpo -tdTomato mice (n = 2), showing tdTomato (red) and CGRP immunostaining (green). Cdx2Flpo-tdTomato+ fibers send collateral terminals to CGRP+ PBel regions as indicated by the arrow, besides projection to PBel and PBdvl. D3V, third ventricular, dorsal division; LHb, lateral habenular nucleus; MC, mediocentral thalamic nucleus; MD, mediodorsal thalamic nucleus; MHb, medial habenular nucleus; mt, mammillothalamic tract; MTh, the medial thalamic nuclei that include MD, MC and MV; MV, medioventral thalamic nucleus; PBdvl, dorsal and ventral subnuclei of parabrachial nucleus; PBel, external lateral parabrachial nucles; PBsl, superior lateral parabrachial nucleus; PVT, paraventricular thalamic nucleus; LHb, lateral habenular nucleus; MTh, medial thalamic nucleus; PVT, paraventricular thalamic nucleus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus. Scale bars: 50 μm in a-c; 100 μm in e-i.

Extended Data Fig. 2

Extended Data Fig. 2. Functional connections of Tac1 _Cre_-derived neurons to neurons in PBsl.

a, Representative images showing the distribution of presynaptic reporter (the synaptophysin-EGFP fusion protein) 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 the potential axons passing 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) 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 were used as control, which are generated by crossing Tac1 Cre and Cdx2 Flpo mice with intersectional GFP reporter mice RC:: FrePe. c, The ascending Tac1Cdx2-CatCh-GFP+ terminals (observed from 3 mice) are detected in the medial thalamic region (left panel), including the paraventricular nucleus (PVT) and the medial thalamic nuclei (MTh). Right panel shows the GFP signals in the superior lateral parabrachial nucleus (PBsl), although the fluorescent signals of CatCh-GFP fusion protein detected by immunostaining are not as robust as by the direct visualization of tdTomato signals observed in Tac1 _Cdx2_-tdTomato mice shown in Figure 1. Scale bars: 100 μm. d, Upper, schematic diagram of optogenetic activation of Tac1Cdx2-CatCh+ terminals in the PBN area via 473 nm blue light, and recording sites for neurons in the PBsl or PBel nucleus of the same brain slices. Lower, 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, 4/15; PBel, 0/15; CHI-test, χ2.95,(1) = 4.615, P = 0.032; Tac1 Cdx2 -CatCh mice, n = 2).

Extended Data Fig. 3

Extended Data Fig. 3. Retrograde labeling of spinal Tac1 _Cre_-derived projection neurons from parabrachial and medial thalamic nuclei and anterograde tracing from the dorsal part of lateral parabrachial nuclei (PBN).

a, Fluorogold retrograde labeling from the PBN of Tac1Cre-tdTomato mice (n = 3). Left, the injection site. Middle and right, a representative transverse section of the dorsal horn showing Fluorogold+ retrograde labeled 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 labeled neuron, showing that _Tac1Cre_-derived neurons represent a subset of spinoparabrachial projection neurons (n = 3, 27.2 ± 0.7%). b, Fluorogold retrograde labeling from the medial thalamic nuclei (MTh, n = 3 mice). Left, the injection site. Large arrowhead indicates that fluorogold injection didn’t leak to the lateral VPM/VPL complex. Middle and right, a representative transverse section of the dorsal horn, showing Fluorogold+ retrograde labeled 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 labeled neurons, indicating that _Tac1Cre_-derived neurons again represent a subset of spinothalamic projection neurons (n = 3 mice, 16.3 ± 3.8%). c, Anterograde tracing from dorsal lateral PBN (including superior lateral and dorsolateral subdivisions, PBsl and PBdvl, respectively). 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 dot circle (arrowhead) indicates the external lateral PBN (PBel) that contains none or little injected tracer. Right, the projection of PBsl-PBdvl neurons to thalamic and hypothalamic regions (Bregma −1.70 mm). Boxes d1, d2 and d3 highlight projections or lack of projections to the medial thalamic nuclei, the ventral lateral thalamic nuclei and the amygdaloid nuclei shown in d, respectively. HY, hypothalamic nuclei; MTh, medial thalamic nuclei. d, Dense innervations were observed in MTh (b1), the lateral habenular nucleus (b1, LHb), and the paraventricular nucleus of thalamus (b1, PVT). No innervations were observed in the medial or lateral parts of the ventral posterolateral nuclei (b2, VPM and VPL), or the central and basal lateral parts of amygdala (b3, CeA and BLA). The lack of innervations to CeA, which is innervated by CGRP+ neurons in PBel, provided further indication that 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 Atlas:

http://connectivity.brain-map.org/projection/experiment/siv/127469566?imageId=127469776&imageType=TWO\_PHOTON,SEGMENTATION&initImage=TWO\_PHOTON&x=18728&y=17591&z=3

. The injection site picture is acquired and modified from image 104 of 140; the thalamic projection picture is acquired and modified from image 71 of 140.

Extended Data Fig. 4

Extended Data Fig. 4. Additional anatomical and behavioral characterizations of Tac1Lbx1 neuron-ablated mice, as well as temporal segregation of withdrawal versus licking responses evoked by noxious heat and their correlation with c-Fos induction in Tac1Cre-tdTomato+ 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 needs 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 system,. b, Representative images showing a marked loss of tdTomato+ cells in the hindbrain spinal trigeminal nucleus (SpV) after DTX injections (n = 3 mice). Arrow in SpV indicates one of few remaining cells and 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 gray nuclei (PAG) or raphe magnus. Aq, aqueduct. c, No detected difference in falling latencies from the rotarod between control littermates and Tac1Lbx1 neuron-ablated mice (“Tac1Lbx1-Abl”) (control, n = 13; Tac1Lbx1-Abl, n = 14; two-sided t-test, P = 0.403). d, No detected difference in response rates to gentle hind paw brushing (out of three tries for each mouse) between control and Tac1Lbx1-Abl groups (control, n = 13; Tac1Lbx1-Abl, n = 14; two-sided Mann-Whitney Rank Sum test, P = 0.121). e, wild type mice showed distinct latencies of lifting/flinching versus licking in response to hot plate stimulation set at 46–47 °C, but no difference at 50 °C (46 °C, n = 10, two-sided paired t-test, P = 0.006; 47 °C, n = 10, two-sided paired t-test, P < 0.001; 50 °C, n = 12, two-sided paired t-test, P = 0.379). In 46 °C hot plate test, licking responses were rarely observed within the first 3 min. f, Representative immunostaining of c-Fos in superficial dorsal horn of Tac1 Cre -tdTomato mice 2 hours after 3-min exposure at the 46 °C or 50 °C hot plate (n = 3 for each condition). Only 50 °C could induce significant amount of c-Fos expression. Lower panels represent the boxed area. Arrows indicate colocalization of c-Fos (green) with Tac1Cre-tdTomato+ cells (red). Scale bars: 100 μm in b, 25 μm in f. Data was presented as mean ± s.e.m. in c, e; and median ± quartile in d.

Extended Data Fig. 5

Extended Data Fig. 5. Tac1Lbx1 neuron-ablated mice still produced licking responses to intraplantar capsaicin injection.

No difference in licking evoked by 10 μl solution containing 3 μg (left) or 0.03 μg (right) capsaicin (3 μg-test: control, n = 15; Tac1Lbx1-Abl, n = 10, two-sided t-test, t(23) = 1.714, P = 0.143; 0.03 μg-test: control, n = 7; Tac1Lbx1-Abl, n = 7, two-sided t-test, t(12) = 0.519, P = 0.613), suggesting the existence of Tac1Lbx1 neuron-independent pain pathways. This preservation of capsaicin-evoked licking is drastically different from a complete loss of licking evoked by mustard oil (MO) and other noxious stimuli (Fig. 3). Licking responses evoked by mustard oil at low concentration (≤ 0.75%) is dependent on TRPA1, and TRPA1 is expressed in a subset of TRPV1+ neurons. As such, MO-responsive neurons only represent a subset of capsaicin-responsive neurons,. In other words, there are capsaicin-sensitive, MO-insensitive, neurons that could in principle mediate Tac1Lbx1 neuron-independent licking evoked by capsaicin. Data shown as mean ± s.e.m..

Extended Data Fig. 6

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

During 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 just experienced. The four sensory qualities were itch, pricking/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/stinging: U = 69.5, P = 0.663; burning: U =71.0, P = 0.723; 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) (upper, continuous pain rating at different time points during the 1-min 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; lower, the areas under the entire curve, “AUC”, again 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 detectable gender differences with the current sample sizes is consistent with previous studies showing that gender differences for experimentally evoked pain are not easy to detect in humans,.

Extended Data Fig. 7

Extended Data Fig. 7. Loss of pinch-induced c-Fos expression in the dorsal horn, the PBsl nucleus, and the lateral habenula, and attenuated pruritogen-induced scratching in Tac1Lbx1 neuron-ablated mice.

a, Representative lumbar spinal cord sections of P60 Tac1 Cre -tdTomato mice (n = 4) after hindpaw pinch stimulation. 41.7 ± 8.0% neurons with pinch-induced c-Fos co-expressed tdTomato. Arrows indicate co-localization and arrowheads indicate singular expression. b, Reduced c-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 c-Fos on coronal sections through the lateral parabrachial nuclei (PBN). Note in wild type littermates, pinch-induced c-Fos was enriched in the superior lateral PBN (PBsl), rarely in external lateral PBN (PBel). Right panel shows quantification of c-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; pinch group: control littermates, n = 8; Tac1Lbx1-Abl, n = 7). 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 level c-Fos expression (no-pinch groups, P = 0.72), an increase in control littermates within the 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 c-Fos induction by pinch, which is in consistent with previous electrophysiological studies,. Right panel shows the counting of pinch-induced c-Fos+ cells in an LHb region adjacent to the MHb from Bregma −1.46 to −2.06 mm (no-pinch groups: control littermates, n = 4; Tac1Lbx1-Abl, n =4; pinch groups: control littermates, n = 7; Tac1Lbx1-Abl, n = 7). 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 c-Fos expression (no-pinch groups, P = 0.289), significant increase in control littermates’ LHb (P = 0.008), and loss of this increase in Tac1Lbx1-Abl mice (P = 0.003). Due to high background c-Fos expression in the paraventricular thalamic nucleus (PVT) and medial thalamic nuclei (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; Tac1Lbx1-Abl: n = 8; two-sided Mann-Whitney Rank Sum test, P = 0.721). f, Reduced scratching bouts induced by intradermal pruritogen injection (compound 48/80: control, n = 15; Tac1Lbx1-Abl, n = 14; two-sided Mann-Whitney Rank Sum test, P = 0.002; chloroquine: control, n = 14; Tac1Lbx1-Abl, n = 14; two-sided Mann-Whitney Rank Sum test, P = 0.005). “Ctrl”: control littermates. n.s.: P > 0.05. b-d, data shown as mean ± s.e.m.; e, f, data shown as mean ± quartile. Scale bars: 50 μm in a and b, 100 μm in c and d.

Extended Data Fig. 8

Extended Data Fig. 8. Loss of pinch-induced CPA in Tac1Lbx1-neuron ablated female mice.

a, A pinched mouse hindpaw. The alligator clip was applied to the ventral skin surface between the footpad and the heel. b, The experimental paradigm 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 of time staying in the paired chamber before (baseline, t1) and after (test, t2) pinch-evoked conditioning. In 3-independent batches of wild type control littermates, 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, p = 0.0979; batch 2, n = 8, P = 0.0097; batch 3, n = 7, P = 0.0002). Two-Way ANOVA analyses of these three batches indicated pinch-evoked avoidance to 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), suggesting that pinch can induce CPA in wild type females. d, Tac1Lbx1 neuron-ablated female mice 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), as male mice did (Fig. 3).

Extended Data Fig. 9

Extended Data Fig. 9. Optogenetic activation of Tac1Cre-derived ascending terminals around the parabrachial nucleus (PBN, a-c) or medial thalamus (d-f).

a-c, Viral infection in lumbar spinal cord Tac1Cre+ neurons and subsequent optogenetic activation of the central terminals in the PBN area. a, The AAV-DIO-ChR2 virus, which drove 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 the PBN (dash line) were visualized by a GFP antibody. These mice are referred to as Tac1Cre-ChR2 mice. Right, immunostaining shows ChR2-EYFP fusion protein expression in the lumbar spinal cord. b, Representative images showing double-color immunostaining, revealing 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 Tac1Cre-ChR2-EYFP+ fibers pass through a region (b1, arrow) lateral to the CGRP+ external lateral PBN (b1, PBel, brown), and terminated densely in the superior lateral PBN (b2, PBsl). c, Representative images showing Blue light stimulation-induced c-Fos expression in PBsl of Tac1Cre-ChR2 mice, with much fewer c-Fos+ neurons following 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 fiber in the region right above the PBN. d-f, Optogenetic experiments for spinal Tac1 neurons projected to medial thalamic nuclei (MD, MC and MV). d,e, Generation of the intersectional Tac1Cdx2-CatCh+ mice was described in Extended Data Figure 2b. The optic fiber was implanted above the medial thalamic complex (left scheme). Neuronal activation by blue light stimulation was indicated by the increase of c-Fos+ cells in Tac1 _Cdx2_-CatCh mice in comparison with Tac1 _Cdx2_-GFP mice, as shown by representative images and quantitative analyses (Tac1Cdx2-CatCh: n = 3; Tac1Cdx2-GFP: n = 3; two-sided t-test, P = 0.011). f, The Tac1 Cdx2 -CatCh mice showed progressive avoidance to the blue light-paired chamber during two 15 min-training trials conducted at two consecutive days (see Methods for detail). Two-way ANOVA plus post hoc Bonferroni’s t-test showed a progressive avoidance to 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).

Extended Data Fig. 10

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 c-Fos expression in dorsal horn Tac1Cre-tdTomato+ neurons.

a, TRPV1+ central terminal-ablated mice showed a dramatic 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 for each group, two-sided Mann-Whitney Rank Sum test, ***P < 0.001). This is stark contrast to subtle, insignificant changes seen in Tac1Lbx1 neuron-ablated mice (Fig. 2e). b, Loss of licking behavior evoked by the 50 °C hot plate stimulation (vehicle injection versus intrathecal capsaicin injection groups, n = 9 for each group; licking episodes within one min, two-sided Mann-Whitney Rank Sum test, ***P < 0.001). c, TRPV1+ central terminal-ablated mice also displayed a dramatic reduction in licking evoked by hindpaw burn injury (n = 8 for each group, licking duration within 30 min after skin burn injury, two-sided t-test, P = 0.001). d, Representative immunostaining images and quantitative analyses showing pinch-evoked c-Fos expression (green) in the dorsal horn of Tac1 Cre -tdTomato mice, with or without chemical ablation of TRPV1+ central terminals (n = 4 for each group). Note a reduction of pinch-induced c-Fos expression in Tac1Cre-tdTomato+ cells after ablation of TRPV1+ central terminals (two-sided t-test, P = 0.044). TRPV1+ nociceptors are necessary for both reflexes (a), and licking (b and c) evoked by noxious heat. Earlier studies showed that those DRG neurons with highest TRPV1 expression (TRPV1highest), representing about 10% of TRPV1+ nociceptors, respond to moderate warm-hot stimulation. Like Mrgprd+ nociceptors, these TRPV1highest neurons innervate exclusively the skin epidermis and their development is dependent on the same transcription factor Runx1,. We therefore speculate that these TRPV1highest neurons may involve with first-line reflexes evoked by noxious heat, raising the possibility that there are different subsets of TRPV1+ nociceptors associated with reflexes versus sustained pain evoked by noxious heat. Future experiments are needed to test this hypothesis.

Fig. 1

Fig. 1. Spinal Tac1 neurons project to medial thalamic and superior lateral parabrachial nuclei.

a, b, Representative sections from P30 (postnatal day 30) Tac1 Cre -tdTomato mice (n = 3), showing tdTomato (red) with Tac1 mRNA (green) or NK1R (green) in superficial dorsal spinal laminae. Arrows indicate co-localization and arrowheads indicate singular expression. Inset in (b) shows a NK1R+/tdTomato+ cell. c-e, Representative coronal thalamic sections (100 μm, Bregma −1.70 mm) from P30 Tac1 Cdx2 -tdTomato mice (n = 3). Arrows in (d) indicate the most medial part of LHb. f, Schematic summary of thalamic projections. g, Representative PBN sections from P30 Tac1 Cdx2 -tdTomato mice (n = 3), showing tdTomato (red) and CGRP immunostaining (green). Note innervations in PBsl (g1). Arrowheads in (g2) indicate fibers passing through the area lateral to PBdvl and CGRP+ PBel. h, Schematic summary of parabrachial projections. D3V, third ventricular, dorsal division; LHb, lateral habenular nucleus; LHbm and LHbl: the medial and lateral parts of LHb, respectively; MC, MD and MV, mediocentral, mediodorsal and medioventral thalamic nuclei, respectively; MHb, medial habenular nucleus; mt, mammillothalamic tract; MTh, the medial thalamic nuclei including MD, MC and MV; PBdvl, PBel and PBsl: the dorsoventral, external and superior lateral PBN, respectivley; PVT, paraventricular thalamic nucleus; scp, superior cerebellar peduncle; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nuclei. Scale bars: 50 μm.

Fig. 2

Fig. 2. Tac1 Lbx1 neurons are dispensable for reflexive-defensive reactions.

a, Tac1Lbx1 neuron-ablated (Tac1Lbx1-Abl) mice showed loss of spinal Tac1Cre-tdTomato+ neurons (control, n = 4, 97 ± 7; Tac1Lbx1-Abl, n = 4, 11 ± 3; P < 0.001). Arrow indicates a remaining cell and arrowhead indicates processes from un-ablated primary afferents. Scale bars: 100 μm. b-e, Reflexive response tests. No significant (n.s.) differences in withdrawal responses to von Frey filament (b, P = 0.09), cold (c, P = 0.07), radiant heat (d, P = 0.44), or hot plate (e, P = 0.11, 0.28, and 0.12 for 47 °C, 50 °C, and 56 °C, respectively) stimulus (control: n=13, 15, and 12 for b-d, respectively, for e, n=10, 12, and 14 for 47 °C, 50 °C, and 56 °C, respectively; Tac1Lbx1-Abl: n=12, 13, and 14 for b-d, respectively, for e, n=9, 15, and 12 for 47 °C, 50 °C, and 56 °C, respectively). f, Comparable jumping evoked by 56 °C hot plate (control and Tac1Lbx1-Abl, n = 12, P = 0.80). g, Foot shock test. Control (n = 9) and Tac1Lbx1-Abl (n =10) groups showed no difference in freezing episodes by three repeated electrical stimulations (“E.S.”) (Two-way ANOWA, P = 0.86), and no difference during recall phases (two-sided t-test: 0.5h, P = 0.10; 48h, p =0.58; mean ± s.e.m). h, Two-plate preference tests. No difference in time spending at the test versus the set temperatures (control: n=11; Tac1Lbx1-Abl: n=10; P = 0.10, 0.33, 0.69, 0.67, and 0.28 for test temperatures at 15 °C, 30 °C, 40 °C, 50 °C, and 0 °C, respectively). a-f, h, two-sided t-test. Data shwon as mean ± s.e.m.

Fig. 3

Fig. 3. Tac1 Lbx1 neurons are required for noxious stimuli-evoked licking and conditioned place aversion (CPA), and their activation drove CPA.

a-b, Tac1Lbx1 neuron-ablated (Tac1Lbx1-Abl) mice lost licking evoked by hot plate (a, control: 47 °C, n=10; 50 °C, n=12; 56 °C, n=14; Tac1Lbx1-Abl: 47 °C, n=9; 50 °C, n=15; 56 °C, n=12, P < 0.001) or cold plate (b, control, n=12; Tac1Lbx1-Abl, n=15, P = 0.002; χ2 test for incidence of mice with licking, control, 9/12; Tac1Lbx1-Abl, 2/15; χ2.95,(1) = 10.5, P = 0.001). c, mustard oil injection. Left, licking time course in wild type mice (n = 8). Right, loss of licking in Tac1Lbx1-Abl mice (control, n=8; Tac1Lbx1-Abl, n=6, P < 0.001). d, Reduced licking evoked by hindpaw burn injury (control, n=6; Tac1Lbx1-Abl, n=5; two-sided t-test, P = 0.001; mean ± s.e.m). e, Skin pinching tests. Left, continuous pain ratings during a one-min period (human subjects, n=25). Right, licking time course in wild type mice (n=10), counted every 5 seconds within a one-min period. f, Loss of pinch-evoked licking in Tac1Lbx1-Abl mice (control and Tac1Lbx1-Abl, n = 8; P < 0.001). g, Hindpaw skin pinch induced CPA in control male mice (sham handling group, n=7; pinched group, n=8; two-sided t-test, P < 0.001; mean ± s.e.m), and CPA loss in Tac1Lbx1-Abl mice (pinched control and Tac1Lbx1-Abl, n=8; two-sided t-test, P < 0.001; mean ± s.e.m). Radiant heat did not generate CPA (control and Tac1Lbx1-Abl, n=6, two-sided Mann-Whitney Rank Sum test, P = 0.234, mean ± quartile). h, Left, intraspinal AAV injection and PBN implantation of the optic fiber in adult Tac1 cre mice. Blue light was on (30 Hz, 20 ms pulse width, 10 mW) whenever a mouse entered compartment “b”. Middle and right, optogenetic activation of terminals in PBN (control RFP mice, n=8; ChR2 mice, n=9) induced both acute avoidance (middle, Two-way ANOVA followed by two-sided post hoc Bonferroni’s t-test, *P = 0.012, **P = 0.002) and CPA 24 hours later (right, two-sided t-test, P < 0.001; mean ± s.e.m). a-c, f, two-sided Mann-Whitney Rank Sum test, data shown as mean ± quartile.

Fig. 4

Fig. 4. TRPV1+, but not Mrgprd+ neurons are required for noxious stimuli-evoked licking.

a, Mrgprd+ and TRPV1+ neurons innervate distinct peripheral targets and spinal laminae. G.I.: gastrointestinal. b, Upper, representative in situ hybridization on DRG sections from three pairs of mice, indicating ablation of Mrgprd+ neurons; lower, representative immunostaining images from 3 pairs of mice, showing ablation of TRPV1+ terminals in superficial spinal lamianes following intrathecal (i.t) capsaicin injection. Scale bars: 100 μm. c, Upper, reduced reflexive responses to von Frey filaments in Mrgprd neuron-ablated (Mrgprd-Abl) mice (control and Abl, n=8, two-sided t-test, P = 0.002), without affecting pinch-evoked licking (control, n=7; Abl, n=8, two-sided t-test, P = 0.150). Lower, TRPV1+ terminal-ablated (TRPV1-Abl) mice (vehicle and capsaicin groups, n=9) showed reduced licking by pinch (two-sided t-test, P < 0.001) or cold plate (Mann-Whitney Rank Sum test for licking episode, P = 0.002; χ2 test for incidence of mice with licking, control, 9/9; TRPV1-Abl, 3/9; χ2.95,(1) = 9, P = 0.003), without affecting withdrawal responses (two-sided t-test; von Frey, P = 0.797; cold plantar test, P = 0.060). d, Summary of two pathways required to drive sustained pain-associated coping behaviors versus reflexive-defensive reactions to external threats. “A” and “B” represents separate primary sensory neurons. “A” includes TRPV1+ neurons required for licking evoked by pinch, noxious cold/heat, and skin burn injury, and “B” includes Mrgprd+ neurons for reflexes evoked by von Frey filaments (for additional discussion, see Extended Data Fig.10). Data was presented as mean ± s.e.m., except cold plate test (median ± quartile).

Similar articles

Cited by

References

    1. Mark VH, Ervin FR & Yakovlev PI Stereotactic thalamotomy III. The verification of anatomical lesion sites in the human thalamus. Archives of neurology 8, 528–538 (1963).
    1. Young RF et al. Gamma Knife thalamotomy for the treatment of persistent pain. Stereotact Funct Neurosurg 64 Suppl 1, 172–181 (1995). - PubMed
    1. Price DD Central neural mechanisms that interrelate sensory and affective dimensions of pain. Mol Interv 2, 392–403, 339, doi:10.1124/mi.2.6.392 (2002). - DOI - PubMed
    1. 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, doi:10.1097/j.pain.0000000000000778 (2017). - DOI - PMC - PubMed
    1. Todd AJ Neuronal circuitry for pain processing in the dorsal horn. Nat Rev Neurosci. 11, 823–836 (2010). - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources