Cellular basis of itch sensation

Abstract

Itch and pain are two distinct sensations. Although our previous study suggested that gastrin-releasing peptide receptor (GRPR) is an itch-specific gene in the spinal cord, a long-standing question of whether there are separate neuronal pathways for itch and pain remains unsettled. We selectively ablated lamina I neurons expressing GRPR in the spinal cord of mice. These mice showed profound scratching deficits in response to all of the itching (pruritogenic) stimuli tested, irrespective of their histamine dependence. In contrast, pain behaviors were unaffected. Our data also suggest that GRPR+ neurons are different from the spinothalamic tract neurons that have been the focus of the debate. Together, the present study suggests that GRPR+ neurons constitute a long-sought labeled line for itch sensation in the spinal cord.

Fig. 1

Selective ablation of GRPR⁺ neurons in the spinal cord. Comparison of molecular expression (C, F, I, L and O; black bars: bombesin-sap group; white bars: blank-sap group) in the superficial spinal cord of mice treated with blank-sap (A, D, G, J, M) and bombesin-sap (B, E, H, K, N). (A-B) GRPR expression in lamina I detected by in situ hybridization (ISH). (C) Approximately 80% of GRPR⁺ neurons were lost in the bombesin-sap group compared to the blank-sap group (** P < 0.01). (D-E) NMUR2 expression in lamina I detected by ISH. (F) The number of NMUR2⁺ neurons was similar between the bombesin-sap and blank-sap groups (P > 0.05). (G-H) NK1 receptor expression in the dorsal spinal cord detected by immunocytochemistry. (I) The density of NK1 signal in lamina I was comparable between the bombesin-sap and blank-sap groups (P > 0.05). (J-K) PKCγ in lamina IIi layer detected by immunocytochemistry. (L) The number of PKCγ⁺ neurons was comparable between groups (P > 0.05). (M-N) Neurotensin expression in Lamina II detected by ISH. (O) The number of neurotensin⁺ neurons was comparable between the bombesin-sap and blank-sap groups (P > 0.05). Scale bar: A, 100 μm (A, B, D, E, G, H, J, K, M, N). Student’s t-test. n = 4~6 for each group. Sap, saporin. Data with error bars represent mean ± SEM.

Fig. 2

Selective ablation of GRPR⁺ neurons abolished scratching behaviors. (A) Histamine-evoked scratching behavior in mice treated with bombesin-sap was almost lost compared with the blank-sap control (500 μg/50 μl, P < 0.001). (B) Compound 48/80-evoked scratching behavior in bombesin-sap-treated mice was almost absent (100 μg/50 μl, P < 0.05). (C) 5-HT-evoked scratching behavior in bombesin-sap-treated mice was largely abolished (10 μg/50 μl, P < 0.001). (D) ET-1-evoked scratching behavior (25 ng/50 μl) in bombesin-sap-treated mice was largely gone (P < 0.001). (E) The PAR2 agonist-evoked scratching behavior in bombesin-sap-treated mice was almost absent (SLIGRL-NH2, 100 μg/50 μl; P < 0.001). (F) Chloroquine-evoked scratching behavior in bombesin-sap-treated mice was also absent (200 μg/50 μl) (P < 0.001). (G) Scratching behavior evoked by DCP was nearly blocked in mice treated with bombesin-sap compared with blank-sap (P < 0.001). Two-way repeated measured analysis of variance (ANOVA). n = 6~9 for each group. Sap, saporin; i.d., intradermal; ET-1, endothelin-1; DCP, diphenylcyclopropenone. Data with error bars represent mean ± SEM.

Fig. 3

Normal pain behaviors in mice treated with bombesin-sap. (A) Mechanical sensitivity in bombesin-sap-treated mice as measured by paw withdrawal threshold upon exposure to von Frey filaments was comparable to mice treated with blank-sap. P > 0.05. (B) The tail-flick latencies in the Randall-Selitto test did not show significant difference between groups. P > 0.05. (C-E) Responses to noxious thermal stimulation measured by the paw withdrawal latency (Hargreaves test, C), the water immersion latency (D), and hot plate test (E) were indistinguishable between groups. P > 0.05. (F) Spontaneous pain responses in first (0~10 min) and second phase (10~60 min) of the formalin test was comparable between mice treated with blank-sap and bombesin-sap. P > 0.05. (G) Spontaneous pain response in mustard oil test was comparable between groups. P > 0.05. (H) Spontaneous pain response induced by intraplantar injection of capsaicin (0.1%) was comparable between groups. P > 0.05. Student’s t-test. n = 6~9 for each group. Sap, saporin. Black bars: the bombesin-sap group; white bars: the blank-sap group. Data with error bars represent mean ± SEM.