Activation of superficial dorsal horn neurons in the mouse by a PAR-2 agonist and 5-HT: potential role in itch

Abstract

Itch, an unpleasant sensation associated with the desire to scratch, is symptomatic of dermatologic and systemic disorders that often resist antihistamine treatment. Histamine-independent itch mediators include serotonin (5-HT) and agonists of the protease-activated receptor-2 (PAR-2). We used behavior, Fos immunohistochemistry, and electrophysiology to investigate if these mediators activate spinal dorsal horn neurons in a manner consistent with itch. Intradermal (i.d.) injection of the PAR-2 agonist SLIGRL-NH(2) in the rostral back evoked bouts of directed hindlimb scratches over 20-30 min. Hindpaw injection of SLIGRL-NH(2) produced Fos staining in superficial dorsal horn which was then targeted for single-unit recording. Small id microinjections of SLIGRL-NH(2) or 5-HT identified responsive single units in the superficial dorsal horn of mice anesthetized with pentobarbital. Thirty-eight units characterized as wide dynamic range, nociceptive specific, or mechanically insensitive exhibited significantly increased firing after i.d. SLIGRL-NH(2) for 9 min, to partial (25%) tachyphylaxis with repeated injection. A majority additionally responded to 5-HT (70%), mustard oil (79%), and capsaicin (71%). Seven units isolated with the 5-HT search stimulus exhibited significant and prolonged responses to 5-HT with tachyphylaxis to repeated injections. The majority also responded to SLIGRL-NH(2), mustard oil, and capsaicin. The prolonged responses of superficial dorsal horn neurons to SLIGRL-NH(2) and 5-HT suggest a role in signaling itch. However, their responsiveness to algogens is inconsistent with itch specificity. Alternatively, such neurons may signal itch, whereas noxious stimulus levels recruit these and a larger population of pruritogen-insensitive cells to signal pain which masks or occludes the itch signal.

Figure 1.

FLI in lumbar superficial dorsal horn after intraplantar microinjection of PAR-2 agonist SLIGRL-NH₂. Photomicrograph of lumbar section showing FLI (black nuclei) after intraplantar microinjection of PAR-2 agonist SLIGRL-NH₂ (50 μg in 5 μl). Arrow in inset shows injection site on ipsilateral hindpaw.

Figure 2.

Excitation of superficial lumbar dorsal horn unit by SLIGRL-NH₂ over a time course consistent with scratching. A, PSTH (1 s bins) of superficial lumbar dorsal horn unit's response to intraplantar id microinjection of SLIGRL-NH₂ (arrow). PSTH (right) shows same unit's response to id capsaicin. Inset shows train of action potentials. Time axis is aligned with graph of scratching shown in B. Note that unit firing increased after injection and persisted for 20 min. B, Graph plots mean number of scratch bouts versus time. ●: Mice received id microinjection of SLIGRL-NH₂ into the nape of the neck at time 0. Bouts of hindlimb scratching directed toward the injection site were counted at 5 min intervals. Scratching peaked at 5 min after injection and persisted for 25 min. Error bars indicate SEM; n = 8. ○: Vehicle (isotonic saline; tested separately in same mice). ▾: Spontaneous scratching in same group of mice. There was a significant difference in mean number of scratch bouts over time between SLIGRL-NH₂-evoked and spontaneous scratching (p < 0.05, ANOVA).

Figure 3.

Example of unit identified by SLIGRL-NH₂ search stimulus. A, PSTH shows successive responses to id SLIGRL-NH₂ (arrows), followed by mechanical and thermal stimuli. Inset, Raw action potentials. B, Continued recording from unit in A shows responses to successive id 5-HT and lack of response to vehicle (saline). C, Responses to mustard oil, capsaicin. D, Recording site in lamina I. E, Arrow shows injection site.

Figure 4.

Mean responses to pruritic and algesic stimuli for superficial dorsal horn units isolated using a PAR-2 agonist search strategy. A, PSTH of averaged response of 20 units to three successive id injections of SLIGRL-NH₂ (50 μg/1 μl) at 10 min intervals. Error bars (gray) indicate SEM. B, Mean responses of six SLIGRL-sensitive units to three successive injections of 5-HT. Inset to right, Histologically recovered recording sites (○) compiled on midlumbar section. C, PSTHs of averaged responses of SLIGRL-NH₂-responsive units to mustard oil (MO), capsaicin, mechanical and thermal stimuli, and id saline (vehicle control).

Figure 5.

Responses of lumbar superficial dorsal horn units to repeated id microinjections of SLIGRL-NH₂. Thin lines plot each individual unit's SA (recorded for 1 min × 10) and response to three repeated id microinjections of SLIGRL-NH₂ at 10 min intervals. Thick black line connecting dots with error bars (SEM) shows mean of 18 units tested. *Mean response to first injection of SLIGRL-NH₂ significantly different from SA (p < 0.05, ANOVA).^#Second and third responses to SLIGRL-NH₂ significantly different from first (p < 0.05); i.e., tachyphylaxis. Third mean response not significantly different from second; second and third responses significantly different from SA.

Figure 6.

Individual example of responses of lamina I neuron isolated by 5-HT search stimulus (format as in Fig. 3). A, Responses to 5-HT (14 mm/1 μl), mechanical and thermal stimuli, and lack of response to saline. B, Successive responses to SLIGRL-NH₂ (same unit as in A). C, Lamina I recording site (top) and intraplantar microinjection site (bottom). D, Responses to id histamine (1%/1 μl), topical mustard oil, and id capsaicin.

Figure 7.

Responses to pruritic and algesic stimuli for superficial dorsal horn units isolated using a 5-HT search strategy (format as in Fig. 4). A, Averaged response of seven units to three successive id injections of 5-HT at 10 min intervals. B, Mean responses of units in A to three successive injections of SLIGRL-NH₂. C, Responses of units in A and B to mustard oil, capsaicin, mechanical and thermal stimuli, and id saline [vehicle (veh) control].

Figure 8.

Responses of lumbar superficial dorsal horn units to repeated id microinjections of 5-HT. Format as in Figure 6. *Mean response to first injection of 5-HT significantly different from SA (p < 0.05, ANOVA). ^#First response to 5-HT significantly different from third (p < 0.05); i.e., tachyphylaxis. Third mean response not significantly different from SA.

Figure 9.

Population coding of itch versus pain. Pruritogens excite primary afferent fibers, which in turn excite WDR, NS, and mechano-insensitive neurons in the superficial dorsal horn that transmit itch (left). Noxious stimulation excites nociceptors to recruit a larger population of pruritogen-insensitive spinal WDR and NS neurons that signal pain and simultaneously mask or occlude itch (right).