A spinal neural circuitry for converting touch to itch sensation

Abstract

Touch and itch sensations are crucial for evoking defensive and emotional responses, and light tactile touch may induce unpleasant itch sensations (mechanical itch or alloknesis). The neural substrate for touch-to-itch conversion in the spinal cord remains elusive. We report that spinal interneurons expressing Tachykinin 2-Cre (Tac2^Cre) receive direct Aβ low threshold mechanoreceptor (LTMR) input and form monosynaptic connections with GRPR neurons. Ablation or inhibition markedly reduces mechanical but not acute chemical itch nor noxious touch information. Chemogenetic inhibition of Tac2^Cre neurons also displays pronounced deficit in chronic dry skin itch, a type of chemical itch in mice. Consistently, ablation of gastrin-releasing peptide receptor (GRPR) neurons, which are essential for transmitting chemical itch, also abolishes mechanical itch. Together, these results suggest that innocuous touch and chemical itch information converge on GRPR neurons and thus map an exquisite spinal circuitry hard-wired for converting innocuous touch to irritating itch.

Fig. 1. Tac2^Cre neurons in the spinal cord are activated by mechanical itch stimulation.

a, c, e, g, i Schematic of mice in free ambulating state (a), i.d. CQ injection (c), soft brushing (e), von Frey hair applied to the hindpaw (g), von Frey hair applied to the hairy skin (i). b, d, f, h, j Representative images of c-Fos expression (green) in the spinal cord of Tac2^tdTom (red) mice corresponding to a, c, e, g, i, respectively. Right: higher magnification of the boxed area. Scale bars, 100 μm (left); 50 μm (right). n = 3 mice per group. k Comparison of the total number of c-Fos positive neurons in laminae IIi and IIIo under different conditions. n = 15 sections from three mice per group. l, m Comparison of the percentage of Tac2^tdTom and c-Fos double positive neurons in c-Fos positive neurons in lamina IIi (l) and IIIo (m) under different conditions. n = 15 sections from three mice per group. one-way ANOVA with Tukey post-hoc, ***p = 0.0001. All data are presented as mean ± s.e.m. and error bars represent s.e.m. Source data are provided as a Source Data file.

Fig. 2. Electrophysiological firing patterns and fiber inputs of Tac2^tdTom neurons.

a, d Schematic of the patch-clamp recording of Tac2^tdTom neurons selected from lamina IIi (a) and lamina IIIo (d). b, e Representative trace of tonic firing pattern (b) and phasic-bursting firing pattern (e) at 40 pA. Rheobase was 20 pA. c Percentages of different type of firing patterns of Tac2^tdTom neurons in lamina IIi. n = 74 neurons. f Percentages of different type of firing patterns of Tac2^tdTom neurons in lamina IIIo. n = 36 neurons. g Representative traces showing one Tac2^tdTom neuron in lamina IIi receiving poly-low threshold-Aδ input. It could not follow 20 μA/2 Hz stimulation. The latency was between 6 and 10 ms. h Percentage of each type of input onto Tac2^tdTom neurons in lamina IIi. n = 50 neurons from 5 mice. i Representative traces showing one Tac2^tdTom neuron in lamina IIIo receiving mono-Aβ input. It followed 20 μA/20 Hz stimulation. The latency was less than 6 ms. j Percentage of each types of input onto Tac2^tdTom neurons in lamina IIIo. n = 46 neurons from 5 mice. Source data are provided as a Source Data file.

Fig. 3. Optostimulation of Tac2 neurons evoked itch-related scratching behavior.

a Schematic of blue light stimulation of Tac2^ChR2 neurons. b, c A snapshot (b) and quantification (c) of blue light-induced scratching behaviors in Tac2^ChR2 mice. One-way ANOVA with Tukey post-hoc, *p = 0.012. n = 6 mice per group. d The effect of i.t. morphine on blue light-induced scratches. Two-way ANOVA with Bonferroni post-hoc, ***p = 0.00001, n = 9 for Tac2^WT/saline, n = 7 for Tac2^WT/morphine, n = 11 for Tac2^ChR2/saline, n = 8 for Tac2^ChR2/morphine. e Blue light-induced scratches decreased after BB-sap treatment. Two-tailed Student’s unpaired t-test, *p = 0.029, n = 7 mice per group. f Double IHC of c-Fos (red) and GFP (green) in the spinal cord of Tac2^ChR2 mice following blue light stimulation. g Higher magnification of the boxed area in f. Arrowheads indicate double-stained c-Fos⁺/Tac2^ChR2+ neurons in f, g. Scale bars, 50 μm in f and 10 μm in g. h Quantification of percentages of Tac2^ChR2/c-Fos double positive neurons in c-Fos positive neurons in laminae IIi and IIIo, respectively. n = 3 mice. All data are presented as means ± s.e.m. and error bars represent s.e.m. Source data are provided as a Source Data file.

Fig. 4. Inhibition of Tac2 neurons in the spinal cord attenuated mechanical itch.

a Schematic of intraspinal injection. b A snapshot of mouse with scratching behavior induced by von Frey filament. c IHC image of mCherry⁺ neurons (red) in the cervical spinal cord of Tac2^Cre mice injected with hM4Di-mCherry virus. All 14 mice with virus injections were subjected to IHC confirmation. Scale bar, 50 μm. d, e Mechanical itch test (d) and CQ itch test (e) after chemogenetic inhibition of Tac2^Cre neurons. (d two-way ANOVA with Bonferroni post-hoc, *p = 0.019, ***p = 0.0001, n = 12 mice for Tac2^WT and n = 14 mice for Tac2^Cre; e unpaired two-tailed Student’s t-test, p = 0.72, n = 11 mice per group, ns not significant). f, g Mechanical itch test (f) and CQ itch test (g) after conditional ablation of Tac2^Cre neurons in the spinal cord. (f two-way ANOVA with Bonferroni post-hoc, **p = 0.002, ***p = 0.0001, n = 12 mice per group; g unpaired two-tailed Student’s t-test, p = 0.78, n = 6 mice per group, ns not significant). h IHC of NKB (green) in the cervical spinal cord of control mice (left) and mice with conditional ablation of Tac2^Cre neurons (right). Scale bar, 100 μm. All data are presented as means ± s.e.m. and error bars represent s.e.m. Source data are provided as a Source Data file.

Fig. 5. Mechanical itch depends on GRPR neurons.

a Representative image of c-Fos IHC (green) in the dorsal horn of Grpr^tdTom (red) mice after von Frey hair stimulation (0.07 g) on the nape skin. Arrowhead indicates c-Fos⁺/ Grpr^{tdTom +} neuron. n = 3 mice. Scale bar, 50 μm. b Quantification of the percentage of Grpr^{tdTom +} /c-Fos⁺ neurons in c-Fos⁺ neurons in laminae I–IIo. n = 3 mice. c, d Mechanical itch test (c) and CQ itch test (d) after 500 ng of BB-sap treatment. (c two-way ANOVA with Bonferroni post-hoc, *p = 0.018, **p = 0.005, ***p = 0.00001, n = 10 mice per group; d unpaired two-tailed Student’s t-test, ***p = 0.00001, n = 10 mice per group). e, f Representative traces showing one Grpr^tdTom neuron cannot follow 20 µA/20 Hz stimulation. g Percentage of each types of inputs onto Grpr^tdTom neurons. Data from five mice, n = 42 neurons. All data are presented as means ± s.e.m. and error bars represent s.e.m. Source data are provided as a Source Data file.

Fig. 6. GRPR neurons form monosynaptic connections with Tac2 neurons.

a, c Representative IHC images of NKB staining (green) in the cervical spinal cord of Tac2^tdTom (red) mice (a) and Grpr^tdTom (red) mice (c). b, d High power images of the boxed areas in a, c, respectively. Scale bars, 20 μm in a, c; 5 μm in b, d. e, f Schematic of intraspinal injection of TVA-EGFP/RVG virus and RV-dG-dsRed virus. g Representative ISH image of Tac2 (blue) in the cervical spinal cord after RVdG injections. Arrowheads indicate Grpr^iCre starter neurons (yellow) expressing GFP (green) and dsRed (red). Arrows indicate Tac2⁺ input neurons (dsRed⁺ and blue⁺) targeting starter neurons. n = 3 mice. Scale bar, 100 μm.

Fig. 7. Tac2 neurons are required for alloknesis and dry skin itch.

a Increased mechanical itch in AEW-treated mice relative to the control. Two-way ANOVA with Bonferroni post-hoc. ***p = 0.00001, n = 10 mice per group. b, c Mechanical itch (b) and spontaneous scratching behavior (c) of AEW-treated mice after chemogenetic inhibition of spinal Tac2^Cre neurons. (b two-way ANOVA with Bonferroni post-hoc, *p = 0.027, **p = 0.005 for group 0.04 g, **p = 0.009 for group 0.16 g, ***p = 0.0001, n = 12 mice per group; c unpaired two-tailed Student’s t-test, **p = 0.001, n = 12 mice per group). d Representative IHC image of c-Fos (green) in the cervical spinal cord of AEW-treated Tac2^tdTom (red) mice. Higher magnification of the boxed area (middle), which is shown at higher magnification (right). Arrowheads indicate double c-Fos⁺/Tac2^tdTom+ neurons. Scale bars, 100 μm in left of d; 50 μm in middle of d; 25 μm in right of d. e Quantification of c-Fos+ neurons in laminae I-IIo, IIi and IIIo, respectively. f The ratio of Tac2^tdTom and c-Fos double positive neurons in c-Fos positive neurons in laminae IIi and IIIo. g Comparison of the number of c-Fos⁺ neurons in laminae I-IIo, IIi and IIIo between control mice stimulated by von Frey hair (0.07 g) and AEW mice. h Comparison of the ratio of c-Fos⁺/ Tac2^tdTom+ neurons in lamina IIi versus IIIo between control and AEW mice. *p = 0.016, unpaired two-tailed Student’s t-test, n = 3 mice per group. All data are presented as means ± s.e.m. and error bars represent s.e.m. Source data are provided as a Source Data file.

Fig. 8. Increased excitability of Tac2^tdTom neurons of dry skin mice.

a–d Resting membrane potential (RMP) (a, b) and the rheobase of action potential (c, d) for Tac2^tdTom neurons in lamina IIi (a, c) and lamina IIIo (b, d). Control: n = 14 neurons in a, b, 13 neurons in c and 12 neurons in d. Dry skin: n = 17 neurons in a, c, 14 neurons in b and 13 neurons in d. Two-tailed Mann–Whitney test. **p = 0.0041, ns not significant. Data were from three mice per group. e Representative traces for Aβ-evoked APs in Tac2^tdTom neurons in laminae IIi of control mice (left) and dry skin mice (right). f, g Percentage of Aβ-evoked APs in Tac2^tdTom neurons in laminae IIi (f) and IIIo (g) of control and dry skin mice, respectively. Control: n = 24 neurons in f, n = 11 neurons in g; dry skin: n = 22 neurons in f, n = 11 neurons in g, Chi-square test, ***p = 0.0001, ns not significant. Data from three mice per group. h Firing patterns of Tac2^tdTom neurons in control mice (n = 24 neurons) and dry skin mice (n = 22 neurons). Data from five mice per group. i Representative traces for Aβ-eIPSCs in Tac2^tdTom neurons in lamina IIi of control mice (left) and dry skin mice (right). j The amplitude of Aβ-eIPSCs on Tac2^tdTom neurons in lamina IIi of control mice and dry skin mice. ***p = 0.00001, two-tailed unpaired Student’s t-test, n = 18 neurons per group. k Representative traces of Aβ-eIPSCs on Tac2^tdTom neurons in lamina IIi before (control) and after bath application of bicuculline (Bic) and/or strychnine (Stry). Blue arrows indicate stimulation artifacts. Data were from three mice per group. l The amplitude of Aβ-eIPSCs on lamina IIi Tac2^tdTom neurons before (control) and after bath application of bicuculline or strychnine. ***p = 0.0001, two-tailed paired Student’s t-test, n = 15 neurons per group. Data are presented as means ± s.e.m. Error bars represent s.e.m. Source data are provided as a Source Data file.

Fig. 9. Schematics showing the spinal neural circuitry for touch-to-itch conversion.

a Under naive condition, Tac2/Ucn3^tdTom neurons in lamina IIIo receive innocuous light touch information via LTMR Aβ/TLR5 fibers directly and in turn relay the touch information to GRPR neurons in laminae I–IIo, which convert it to itch. By contrast, Tac2/Ucn3^tdTom neurons in lamina IIi which receive direct inputs from C/Aδ fibers and indirect Aβ inputs may remain silent or inactive resulting from feedforward inhibition of GABAergic/glycinergic neurons. b Under dry skin itch condition, GRP primary afferents convey chemical itch information via GRP to GRPR neurons directly. In addition, enhanced chemical itch relayed by C/Aδ fibers recruit IIi Tac2/Ucn3^tdTom neurons to relay chemical itch information to GRPR neurons. Light touch information conveyed by Tac2/Ucn3^tdTom neurons, along with newly recruited IIi Tac2/Ucn3^tdTom neurons, and chemical itch converge on GRPR neurons. Augmented activity of GRPR function can also be resultant from reduced or a loss of feedforward inhibition mediated by GABAergic/glycinergic neurons, giving rise to exacerbated chronic itch and alloknesis.