GRP receptor and AMPA receptor cooperatively regulate itch-responsive neurons in the spinal dorsal horn

Abstract

Gastrin-releasing peptide (GRP) receptor-expressing (GRPR)⁺ neurons have a central role in the spinal transmission of itch. Because their fundamental regulatory mechanisms are not yet understood, it is important to determine how such neurons are excited and integrate itch sensation. In this study, we investigated the mechanisms for the activation of itch-responsive GRPR⁺ neurons in the spinal dorsal horn (SDH). GRPR⁺ neurons expressed the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) containing the GluR2 subunit. In mice, peripherally elicited histaminergic and non-histaminergic itch was prevented by intrathecal (i.t.) administration of the AMPAR antagonist NBQX, which was consistent with the fact that firing of GRPR⁺ neurons in SDH under histaminergic and non-histaminergic itch was completely blocked by NBQX, but not by the GRPR antagonist RC-3095. Because GRP⁺ neurons in SDH contain glutamate, we investigated the role of GRP⁺ (GRP⁺/Glu⁺) neurons in regulating itch. Chemogenetic inhibition of GRP⁺ neurons suppressed both histaminergic and non-histaminergic itch without affecting the mechanical pain threshold. In nonhuman primates, i.t. administration of NBQX also attenuated peripherally elicited itch without affecting the thermal pain threshold. In a mouse model of diphenylcyclopropenone (DCP)-induced contact dermatitis, GRP, GRPR, and AMPAR subunits were upregulated in SDH. DCP-induced itch was prevented by either silencing GRP⁺ neurons or ablation of GRPR⁺ neurons. Altogether, these findings demonstrate that GRP and glutamate cooperatively regulate GRPR⁺ AMPAR⁺ neurons in SDH, mediating itch sensation. GRP-GRPR and the glutamate-AMPAR system may play pivotal roles in the spinal transmission of itch in rodents and nonhuman primates.

Keywords: Chloroquine; Glutamate; Histamine; Nonhuman primate; Pain; Pruritus.

Fig. 1.

Localization of AMPAR on GRPR⁺ neurons in the spinal dorsal horn (SDH) and itch regulation. (A) GRPR antagonist, RC-3095 (0.1 nmol) or vehicle was intrathecally (i.t.) administered 10 min prior to intradermal (i.d.) injection of pruritogens. (B) Bombesin-saporin (Bom-Sap, 250 ng), GRPR⁺ cell-targeting toxin, or blank-saporin (Sap, 250 ng) was administered i.t. 2 weeks before i.d. injection of pruritogens. (A, B, E) Non-histaminergic pruritogens [chloroquine (300 nmol) and SLIGRL (100 nmol)] and histaminergic pruritogens [compound 48/80 (300 nmol) and HTMT (300 nmol)] were used. Scratching bouts were observed immediately after i.d. administration up to 30 min. (A, B) Total number of scratching bouts for 30 min are shown. n = 5. ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 vs vehicle or Sap. (C) The mRNA expression levels of GRPR, NK1R, and AMPAR subunits (GluR1–4) in the lumbar SDH at 2 weeks after i.t. administration of Bom-Sap (250 ng) or blank-Sap (250 ng) were evaluated by RT-qPCR. n = 5–8. ***P < 0.001, *P < 0.05 vs Sap. (D) Protein expression of GRPR and GluR2 in the lumbar SDH were visualized by immunohistochemistry in naïve mice. Representative micrographs from four mice are shown. Scale bars = 100 μm. (E) AMPAR antagonist, NBQX (0.1, 0.3 nmol), or vehicle was i.t. administered 10 min prior to i.d. injection of pruritogens. Time course in 10-min intervals (left) and total number of scratching bouts for 30 min are shown (right). n = 5. ^##P < 0.01, ^#P < 0.05 vs vehicle. (A–C, E) Each value represents the mean ± S.E.M.

Fig. 2.

AMPAR is critical for peripherally evoked firing of GRPR⁺ neurons in the SDH. Spontaneous firing in the absence and presence of GRP (500 nM) in Krebs solution obtained from lumbar SDH neurons using in vivo extracellular recording. (A) Responsiveness of GRP application on spontaneous firing from 50 SDH neurons. (B) Percentage of GRP-responsive neurons. (C) Summary of recording depth on GRP-responsive and nonresponsive neurons. (A–C) n = 12–50. ***P < 0.001 vs baseline. Representative traces (D, F) and summaries (E, G) showing the effects of RC-3095 (3 μM) and NBQX (20 μM) on compound 48/80-(300 nmol) or chloroquine- (300 nmol) evoked firing of GRP-responsive SDH neurons. n = 5–6. ***P < 0.001 vs baseline. ^##P < 0.01 vs control. (A–C, E, G) Each value represents the mean ± S.E.M.

Fig. 3.

Expression of the Cre-dependent fluorescence and Gi-DREADD system on GRP⁺ neurons in the SDH. (A) R26-LSL-tdTomato mice were crossed with GRP-Cre mice for generation of GRP-tdTomato mice. (B) Cre-dependent expression of tdTomato in the SDH of GRP-tdTomato mice but not ROSA26 heterozygous mice. The protein expression of GRP was visualized by immunohistochemistry. (C) R26-LSL-Gi-DREADD mice were crossed with GRP-Cre mice for generation of GRP-Gi-DREADD mice. (D) The Cre-dependent expression of HA-tagged hM4Di in the SDH of GRP-Gi-DREADD mice but not of ROSA26 heterozygous mice was visualized by immunohistochemistry. (E) Double immunostaining of HA, GRP, TRPV1, VGLUT2, or IB4-labeled non-peptidergic C-fibers in the SDH of GRP-Gi-DREADD mice. Representative micrographs from four mice are shown. Scale bars = 100 μm.

Fig. 4.

Chemogenetic silencing of GRP⁺ neurons in the SDH prevents peripherally elicited itch. Clozapine-N-oxide (CNO) was intraperitoneally (A, C; i.p., 0.3 mg/kg) or i.t. (B; 3 nmol) administered 40 min prior to i.d. injection of pruritogens in GRP-Gi-DREADD mice. Non-histaminergic pruritogens [chloroquine (300 nmol), SLIGRL (100 nmol), and BAM8–22 (50 nmol)] and histaminergic pruritogens [compound 48/80 (300 nmol) and HTMT (300 nmol)] were used. Scratching bouts were observed immediately after i.d. administration up to 30 min. Time course in 10-min intervals (left) and total number of scratching bouts for 30 min are shown (right). n = 6–7. ^#P < 0.05 vs vehicle. (C) Chloroquine (300 nmol) was i.d. administered to the right calf of GRP-Gi-DREADD mice, and the protein expression of c-fos in the lumbar SDH at 2 h after administration was visualized by immunohistochemistry. Representative micrographs and mean number of c-fos⁺ cells in each group are shown. n = 6. ***P < 0.001 vs contralateral/vehicle. ^#P < 0.05 vs ipsilateral/vehicle. (A–C) Each value represents the mean ± S.E.M.

Fig. 5.

Behavioral effects of intrathecal administration of AMPAR antagonist NBQX in monkeys. (A) Effects on peripherally elicited itch. NBQX (1, 3 nmol) was i.t. administered 30 min prior to i.d. administration of BAM8–22 (50 nmol). Scratching was recorded 1 min after i.d. administration up to 16 min. Total number of injection site-specific scratching for 1–16 min is shown. Each value represents the mean ± S.E.M. n = 5. *P < 0.05 vs vehicle. (B) Effects on thermal nociception. Temperature-response curves were measured by using the tail-withdrawal latency at 30 min after i.t. administration of NBQX (3 nmol). Each value represents the mean ± S.E.M. n = 4.

Fig. 6.

Roles of GRP-GRPR and glutamate-AMPAR systems in pathological itch by contact dermatitis. (A) Schedule of the diphenylcyclopropenone (DCP) application and photo of the acetone- or DCP-treated mice on day 7. (B, F, G) Scratching bouts were observed immediately after DCP application up to 40 min, and the total number of scratching bouts are shown. (B) Time course of DCP-induced scratching behavior. The mRNA expression levels of GRP and GRPR (C) on indicated days and AMPAR subunits (GluR1–4; E) on day 14 in the cervical SDH were evaluated by RT-qPCR. n = 6. ***P < 0.001, **P < 0.01, *P < 0.05 vs acetone. (D) Protein expression of GRR and GRPR in the cervical SDH on day 14 were visualized by immunohistochemistry. Representative micrographs from four mice are shown. Scale bars = 100 μm. (F) Inhibition of DCP-induced scratching behavior by CNO administration (0.3 mg.kg, i.p., 30 min prior to DCP) in GRP-Gi-DREADD mice. (G) Prevention of DCP-induced scratching behavior in mice after Bom-Sap treatment (250 ng, i.t., 7 days before experiment). n = 8–9. ^#P < 0.05 vs control or saporin. E–G) Each value represents the mean ± S.E.M. (H) Schematic spinal transmission of itch. Under physiological and pathological condition, GRP and glutamate released from SDH neurons activate itch-responsive spinal neurons through GRPR and/or AMPAR.