Identification of spinal circuits involved in touch-evoked dynamic mechanical pain

Abstract

Mechanical hypersensitivity is a debilitating symptom for millions of chronic pain patients. It exists in distinct forms, including brush-evoked dynamic and filament-evoked punctate hypersensitivities. We reduced dynamic mechanical hypersensitivity induced by nerve injury or inflammation in mice by ablating a group of adult spinal neurons defined by developmental co-expression of VGLUT3 and Lbx1 (VT3^Lbx1 neurons): the mice lost brush-evoked nocifensive responses and conditional place aversion. Electrophysiological recordings show that VT3^Lbx1 neurons form morphine-resistant polysynaptic pathways relaying inputs from low-threshold Aβ mechanoreceptors to lamina I output neurons. The subset of somatostatin-lineage neurons preserved in VT3^Lbx1-neuron-ablated mice is largely sufficient to mediate morphine-sensitive and morphine-resistant forms of von Frey filament-evoked punctate mechanical hypersensitivity. Furthermore, acute silencing of VT3^Lbx1 neurons attenuated pre-established dynamic mechanical hypersensitivity induced by nerve injury, suggesting that these neurons may be a cellular target for treating this form of neuropathic pain.

Figure 1

Characterization of VT3^Cre-tdTomato neurons and punctate sensitivity in VT3^Lbx1 neuron-ablated mice. (a-c) Spinal sections from P4 mice (a) and adult mice (b,c) showing tdTomato signals (red) and VGLUT3 mRNA, NK1R protein, IB4 isolectin binding, PKCγ protein, or VGLUT2 mRNA (green). Right panels in a represent higher magnification of the boxed areas. Arrows indicate co-localization, and arrowhead in a indicates a cell expressing VGLUT3 mRNA but having not yet activated tdTomato. (d) The intersectional genetic strategy for driving DTR expression in spinal VT3^Lbx1 neurons. (e) Ablation of 86% of VT3^Cre-tdTomato⁺ neurons in adult lumbar cord (Control: 96 ± 11 per hemisection, Ablated (“VT3^Cre-Abl”) mice:14 ± 4; n = 3 mice per group; two-tailed student’s unpaired t test; t₄ = 6.885, ***, P = 0.002). Large arrowhead indicates remained cells, and small arrowhead indicates processes likely from VT3^Cre-tdTomato⁺ primary afferents. (f) Increased withdrawal thresholds to von Frey filament stimulations (n = 17, Control; n = 15, Ablated; two-tailed student’s unpaired t test; t₃₀ = 4.4107, **, P < 0.01). No detectable changes in pinprick responses (n = 10, Control; n = 12, Ablated; unpaired t test, t₂₀ = 1.180, P = 0.2519), pinch (n = 10, Control; n = 12, Ablated; two-tailed student’s unpaired t test, t₂₀ = 0.7881, P = 0.4399), or Randall-Sellito (n = 11 in each group; two-tailed student’s unpaired t test, t₂₀ = 1.618, P = 0.1213). Scale bars are 50 μm in all images. Data are represented as mean ± SEM.

Figure 2

Loss of dynamic mechanical hypersensitivity in VT3^Lbx1 neuron-ablated (“VT3^Lbx1 Abl”) mice. (a) Schematics for dynamic and punctate stimulations. (b-e) Loss of brush-evoked dynamic hypersensitivity in VT3^Lbx1 Abl mice following SNI (b, left, n = 13, Control; n = 11, VT3^Lbx1 Abl; two-way ANOVA, F_{6, 154} = 4.783, P = 0.0002), CFA (c, left, n = 5, control; n = 6, VT3^Lbx1 Abl; two-way ANOVA, F_{1, 24} = 12.37, P = 0.0018), 3% carrageenan (d, top left, n = 7, control; n = 6, VT3^Lbx1 Abl; two-tailed student’s unpaired t test, t₁₁ = 5.730, P = 0.0001), or following Intrathecal injection of bicuculline and strychnine (”Bic+Stry”) (e, left, n = 6, Control; n = 6, VT3^Lbx1 Abl; two-way ANOVA, F_{6, 70} = 14.0925, P < 0.0001). No changes in punctate hypersensitivity after SNI (b, right, n = 13, Control; n = 11, VT3^Lbx1 Abl; before SNI, two-tailed student’s unpaired t test, t₂₂ = 3.798, P = 0.0010; after SNI, two-way ANOVA, F_{5, 132} = 0.6077, P = 0.6941), CFA (c, right, n = 5, Control; n = 6, VT3^Lbx1 Abl; before CFA, two-tailed student’s unpaired t test, t₉ = 2.922, P = 0.0119; after CFA, two-way ANOVA, F_{1, 24} = 2.391, P = 0.1351), 3% carrageenan assay (d, top right, n = 7, Control; n = 6, VT3^Lbx1 Abl; before carrageenan, two-tailed unpaired t test; before carrageenan, t₁₁ = 10.28, P < 0.0001; after carrageenan, two-tailed unpaired t test, t₁₁ = 0.2360, P = 0.8178), or following Bic+Stry (e, right, n = 6, Control; n = 6, VT3^Lbx1 Abl; before Bic+Stry, two-tailed student’s unpaired t test, t₁₀ = 5.811, P = 0.0002; after Bic+Stry, two-way ANOVA, F_{1, 60} = 1.260, P = 0.2662). (d, bottom) 0.5% carrageenan treatment. An increase in withdrawal thresholds before inflammation (n =12, control; n =16, VT3^Lbx1 Abl; two-tailed student’s unpaired t test, t₂₆ = −5.174, P < 0.0001). VT3^Lbx1 neuron-ablated mice can be divided into two clusters after inflammation. Cluster 1 (10/16) showed withdrawal thresholds (0.029 ± 0.013g; n = 10) indistinguishable from control mice (0.025 ± 0.028 g; n =12; two-tailed student’s unpaired t test, t₂₀ = −0.377, P = 0.7100). Cluster 2 (6/16), however, showed withdrawal thresholds (0.96 ± 0.89 g; n =6) comparable to that seen in ablation mice without inflammation (1.6 ± 0.80; n =16; two-tailed student’s unpaired t test, t₂₀ = 1.531, P = 0.1414), and significantly higher than that in control mice (0.025 ± 0.028 g; n =12; two-tailed student’s unpaired t test, t₁₆ = −3.74, P = 0.0018). Data are represented as mean ± SEM. ***, P < 0.001.

Figure 3

Reduction of brush-evoked c-Fos in VT3^Lbx1 neuron-ablated (“VT3^Lbx1-Abl”) mice and attenuation of dynamic mechanical hypersensitivity by silencing VT3^Lbx1 neurons. (a) c-Fos induction after brushing the hindpaw of control mice (with or without SNI) or VT3^Lbx1-Abl mice (with SNI) (n = 3 mice per group; two-tailed student’s unpaired t test; t₄ = 4.260, *, P = 0.0131). Scale bars are 100 μm in the left images and 50 μm in the right. (b) Schematics showing the intersectional genetic strategy to drive hM4Di in VT3^Lbx1 neurons. (c,d) Impact of VT3^Lbx1 neuron silencing via CNO-mediated activation of hM4Di on mechanical hypersensitivity (n = 6, Control; n = 6, VT3^Lbx1-hM4Di). Left columns: attenuation of dynamic hypersensitivity at day 7 post SNI (c left, two-tailed student’s unpaired t test, 40min post-CNO, t₁₀ = 3.3, P = 0.0080; 50min post-CNO, t₁₀ = 3.789, P = 0.0035) or at day 30 post SNI (d, two-tailed student’s unpaired t test, 40min post-CNO, t₁₀ = 4.287, P = 0.0016; 50min post-CNO, t₁₀ = 3.501, P = 0.0057). Right columns: no detectable change in punctate hypersensitivity at day 7 post SNI (two-tailed student’s unpaired t test, 40min post-CNO, t₁₀ = 0.1638, P = 0.8732; 50min post-CNO, t₁₀ = 0.0702, P = 0.9454) and at day 30 post SNI (two-tailed student’s unpaired t test, 40min post-CNO, t₁₀ = 0.2666, P = 0.7952; 50min post-CNO, t₁₀ = 0.2666, P = 0.7952). Data are represented as mean ± SEM. *, P < 0.05, **, P < 0.01.

Figure 4

Ablation of VT3^Lbx1 neurons suppressed brush-evoked conditioned place aversion (CPA) in mice with nerve injury (SNI). (a) Schematics of the CPA apparatus and the experimental design. The dark and bright chambers are labeled as “A” and “B”, respectively. At day 1 and day 6, the residence times in the dark A chamber during a 15-min period were determined. On days 2–5, the mouse was placed in the indicated chamber for 20 min, with or without brushing. (b,c) Absolute time (s) in the dark A chamber before (“pre”) versus after (“post”) conditioning for various experimental groups (b), or CPA scores defined by the time difference staying in the A chamber: (time before training) – (time after training) (c). n = 6, control littermates without SNI (“naïve”); n = 8, control littermates with SNI; n = 9, VT3^Lbx1 Abl with SNI. For (b), two-tailed student’s paired t test; naive: t₁₀ = 0.3487, P = 0.7345; control-SNI: t₁₄ = 7.249, P < 0.0001; VT3^Lbx1 ablated-SNI: t₁₆ = 1.410, _P = 0.1777; for (c), two-tailed student’s unpaired t test; t₁₅ = 5.066, ***, P = 0.0001. Data are represented as mean ± SEM.

Figure 5

Characterization of type1 and type 2 VT3^Cre-tdTomato⁺ neurons. (a,b) Low threshold Aβ intensity stimulation-induced-inputs/outputs in type 1 and type 2 VT3^Cre-tdTomato⁺ neurons before (a) and after (b) treatment with bicuculline (10 μM) plus strychnine (2 μM). The light gray traces are higher magnifications of the portions of the top black traces marked with the rectangle bar (only shown in the first panel). For type 1 neurons, with the presence of bicuculline and strychnine (“Bic+Stry”), Aβ intensity stimulation evoked both fast-onset (red arrows) and slow-onset (red arrowheads) currents with AP firing. Type 2 neurons (right panel) received slow Aβ inputs only with bicuculline and strychnine. (c) Bath application of NMDAR antagonist APV (50 μM) blocked slow-onset long-lasting Aβ-evoked currents and AP firing induced by bicuculline and strychnine. Black arrows indicate stimulation artifacts, red vertical dashed lines indicate the 10-ms time point following stimulation, and red horizontal dashed lines indicate baseline. (d) Predominant AP firing patterns of type1 and type 2 VT3^Cre-tdTomato⁺ neurons, respectively. All recordings are composed of three repeated traces.

Figure 6

Slow-onset and fast-onset Aβ pathways opened by nerve injury and mediated differentially via VT3^Lbx1 and SOM^Lbx1 neurons. (a-c) Low threshold Aβ intensity-evoked-inputs/outputs in spinal I-II_o neurons in control, VT3^Lbx1-neuron-ablated mice (“VT3^Lbx1 Abl”) and SOM^Lbx1-neuron-ablated mice (“SOM^Lbx1 Abl”), with spared nerve injury (“SNI”) or without (“naïve”). Left: Aβ inputs tested by Aβ-eEPSCs at −70 mV; Right: Aβ outputs tested by Aβ-evoked EPSPs/APs at resting membrane potentials. Black arrows indicate stimulation artifacts, red vertical dashed lines indicate the 10-ms time point following stimulation, and red horizontal dashed lines indicate baseline. All recordings are composed of three repeated traces.

Figure 7

Differential morphine sensitivity of spinal pathways. (a,b) Effects on punctate and dynamic hypersensitivity by intrathecal injection of morphine or saline (n = 6 in each group). In (a), morphine attenuated punctate hypersensitivity at day 7 post SNI in both control and ablation (“VT3^Lbx1 Abl”) mice (two-way ANOVA, F_{1, 10} = 47, P < 0.001) and the degrees of inhibition are no different (two-way ANOVA, F_{1, 10} = 0.0691, P = 0.798). Morphine had no inhibitory effect at day 30 (two-way ANOVA, F_{1, 10} = 0.237, P = 0.637). In (b), morphine did not affect dynamic hypersensitivity in either wild type or VT3^Lbx1 Abl mice (two-way ANOVA, Day 7: F_{1, 10} = 1.403, P = 0.264; Day 30: F_{1, 10} = 0.366, P = 0.558). Note that dynamic hypersensitivity was attenuated in VT3^Lbx1 neuron-ablated mice in comparison with control at either time point (two-way ANOVA; Day 7: F_{1, 10} = 81.824, P < 0.001; Day 30: F_{1, 10} = 60.324, P < 0.001). Data are represented as mean ± SEM. (c) Left panel: schematics illustration of sagittal spinal cord slice preparations to preserve electrically low-threshold Aβ-fiber inputs. Right panel: morphine resistance of Aβ-evoked AP outputs in slice from mice with SNI or with bicuculline plus strychnine (“Bic+Stry”). Red lines indicate the 10-ms time point following stimulation. Black arrows indicate stimulation artifact. Red arrows and arrowheads indicate fast-onset and slow-onset Aβ-evoked APs, respectively. (d) Left panel: in this “C input” preparation, electrically low-threshold Aβ inputs to VT3^Cre-tdTomato-negative _vII_i neurons were absent (left, top two traces). C intensity stimulation-evoked EPSPs and IPSPs without APs (left, bottom trace). Right panel: C intensity stimulation-generated APs outputs with Bic+Stry (d, right panel, “Control”) in VT3^Cre-tdTomato-negative neurons in _vII_i, which were either sensitive or resistant to morphine treatment. All recordings are composed of three repeated traces.

Figure 8

Loss of C-fiber inputs to _vII_i neurons in SOM^Lbx1 neuron-ablated mice (“SOM^Lbx1 Abl”), but not in VT3^Lbx1 neuron-ablated mice (“VT3^Lbx1 Abl”). (a) Schematics showing sagittal spinal cord slice preparations that removed low-threshold Aβ-fiber inputs to dorsal horn neurons. (b–d) C intensity stimulation-induced-inputs/outputs in _vII_i neurons in control, SOM^Lbx1 Abl and VT3^Lbx1 Abl mice. Blue and red arrows indicate C fiber inputs and electrically high threshold (“HT”) Aβ inputs/outputs, respectively. Typical traces shown are representative responses of 23, 37 and 20 neurons, respectively, in control, SOM^Lbx1 Abl, and VT3^Lbx1 Abl mice. All recordings are composed of three repeated traces.