Scratching behavior and Fos expression in superficial dorsal horn elicited by protease-activated receptor agonists and other itch mediators in mice

Abstract

Protease-activated receptor (PAR)-2 and PAR-4 are implicated in nonhistaminergic itch. We investigated dose dependence, tachyphylaxis, and cross-tachyphylaxis of itch-associated scratching elicited by intradermal injections of PAR-2 and PAR-4 agonists, serotonin (5-hydroxytryptamine, 5-HT), and histamine in ICR mice, as well as mu-opioid modulation of PAR-2 agonist-evoked scratching. Each agent elicited dose-related increases in scratch bouts. Scratching elicited by the PAR-4 agonist and histamine both exhibited significant tachyphylaxis but no cross-tachyphylaxis with each other. Scratching evoked by 5-HT did not exhibit significant tachyphylaxis but did exhibit significant cross-tachyphylaxis to scratching evoked by the PAR-2 and PAR-4 agonists and histamine. Naltrexone and high-dose morphine (10 mg/kg) attenuated PAR-2 agonist-evoked scratching, whereas lower dose morphine (1 mg/kg) had no effect. High-dose morphine also significantly increased circling behavior, which may have interfered with scratching. The PAR-2 agonist and 5-HT produced overlapping distributions of Fos-like immunoreactivity in the superficial dorsal horn. These results indicate that PAR-2 and PAR-4 agonists, histamine, and 5-HT elicit itch-related scratching and activate superficial dorsal horn neurons that may participate in scratch reflex and ascending itch signaling pathways.

Fig. 1.

Dose-dependent scratching elicited by various itch mediators. Graph plots mean number of scratch bouts/60 min versus dose of PAR-2 agonist SLIGRL-NH₂, PAR-4 agonist AYPGKF-NH₂, 5-HT, and histamine. Total injected dose calculated as nanomoles in a 10-μl volume (n = 6–8/group). There was a significant overall effect of dose for each agent (ANOVA; p < 0.05). Error bars are S.E.M.

Fig. 2.

Time course, tachyphylaxis, and cross-tachyphylaxis for hind limb scratching elicited by intradermal microinjection of PAR-2 agonist. In each panel, the PAR-2 agonist was injected first, and scratch bouts (□) were counted over 40 min, followed by injection of a second agent (filled symbols), and scratch bouts were counted again over 40 min. A, graph plots mean number of scratch bouts/5-min intervals after first injection of PAR-2 agonist SLIGRL-NH₂ (□; trial 1; 50 μg/10 μl) and after second injection of the same dose of SLIGRL-NH₂ at the same site (▪; trial 2). B, as in A for PAR-2 agonist followed by PAR-4 agonist AYPGKF-NH₂ (50 μg/10 μl). C, as in A for PAR-2 agonist followed by 5-HT (47 mM/10 μl). D, as in A for PAR-2 agonist followed by histamine (50 μg/10 μl). Error bars are S.E.M. (n = 5–8/group).

Fig. 3.

PAR-4 agonist. Graphs as in Fig. 2 for experiments in which the PAR-4 agonist AYPGKF-NH₂ was injected first, followed 40 min later by injection of either the PAR-4 agonist again (A), the PAR-2 agonist (B), histamine (C), or 5-HT (D).

Fig. 4.

Histamine. Graphs as in Fig. 2 for experiments in which histamine was injected first, followed 40 min later by injection of either histamine again (A), the PAR-2 agonist (B), the PAR-4 agonist (C), or 5-HT (D).

Fig. 5.

5-HT. Graphs as in Figs. 2, 3, 4 for experiments in which 5-HT was injected first, followed 40 min later by injection of either 5-HT again (A), the PAR-2 agonist (B), the PAR-4 agonist (C), or histamine (D).

Fig. 6.

Summary of tachyphylaxis and cross-tachyphylaxis results. A, bar graph plots mean number of scratch bouts/40 min elicited by intradermal microinjection of the PAR-2 agonist when it was given first (left-hand bar) and when it was injected 40 min after a prior injection of the same PAR-2 agonist, the PAR-4 agonist, histamine, or 5-HT (second–fifth bars to right). *, p < 0.05, significantly different from first trial of PAR-2 agonist. B, as in A for PAR-4 agonist. *, p < 0.05, significantly different from first trial of PAR-4 agonist. C, as in A for scratch bouts elicited by histamine. *, p < 0.05, significantly different from first trial of histamine. D, as in A for 5-HT. *, p < 0.05, significantly different from first trial of 5-HT.

Fig. 7.

Opioid modulation of PAR-2 agonist-evoked scratching. A, bar graph plots mean number of scratch bouts/45 min. First three bars show, from left to right, number of scratch bouts evoked PAR-2 agonist (SLIGRL-NH₂;50 μg/10 μl) when preceded by intraperitoneal saline (▪; control), morphine (1 mg/kg; ), or naltrexone (1 mg/kg; □), respectively. *, p < 0.01, significant difference between saline and naltrexone groups (n = 5–6/group). Bars to right show scratching elicited by intradermal capsaicin (10 μg/10 μl; ) and lack of significant effect of naltrexone (). B, graph plots total number of scratch bouts/45 min elicited by the PAR-2 agonist SLIGRL-NH₂ versus dose of morphine. The number of scratch bouts at 1- and 3-mg/kg doses of morphine was not significantly different from vehicle but was significantly lower (*, p < 0.05) at the highest morphine dose (10 mg/kg). C, graph plots mean number of rotations (circling)/45 min versus dose of morphine. *, p < 0.05 for all, significantly different from 0-, 1-, and 3-mg/kg doses.

Fig. 8.

Overlapping distributions of FLI in cervical spinal cord after intradermal injections of SLIGRL-NH₂ (50 μg/5 μl) or 5-HT (47 nmol/5 μl) in nape of neck. A, photomicrograph of midcervical section showing FLI elicited by PAR-2 agonist. B, drawing of cervical dorsal horn compiling FLI from one section each from mice injected with PAR-2 agonist (•) or 5-HT (▴) in nape of neck. C, mean counts of FLI from mice receiving intradermal PAR-2 agonist (blue bar; n = 6), 5-HT (red bar; n = 6), or saline (white bar; n = 4). *, p < 0.05 significantly different versus groups receiving PAR-2 agonist and 5-HT (counts for PAR-2 agonist versus 5-HT not significantly different).