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Endothelin-1 (ET-1) has been implicated in nonhistaminergic itch. Here we used electrophysiological methods to investigate whether mouse superficial dorsal horn neurons respond to intradermal (id) injection of ET-1 and whether ET-1-sensitive neurons additionally respond to other pruritic and algesic stimuli or spinal superfusion of bombesin, a homolog of gastrin-releasing peptide (GRP) that excites spinal itch-signaling neurons. Single-unit recordings were made from lumbar dorsal horn neurons in pentobarbital-anesthetized C57BL/6 mice. We searched for units that exhibited elevated firing after id injection of ET-1 (1 μg/μl). Responsive units were further tested with mechanical stimuli, bombesin (spinal superfusion, 200 μg·ml−1·min−1), heating, cooling, and additional chemicals [histamine, chloroquine, allyl isothiocyanate (AITC), capsaicin]. Of 40 ET-1-responsive units, 48% responded to brush and pinch [wide dynamic range (WDR)] and 52% to pinch only [high threshold (HT)]. Ninety-three percent responded to noxious heat, 50% to cooling, and >70% to histamine, chloroquine, AITC, and capsaicin. Fifty-seven percent responded to bombesin, suggesting that they participate in spinal itch transmission. That most ET-1-sensitive spinal neurons also responded to pruritic and algesic stimuli is consistent with previous studies of pruritogen-responsive dorsal horn neurons. We previously hypothesized that pruritogen-sensitive neurons signal itch. The observation that ET-1 activates nociceptive neurons suggests that both itch and pain signals may be generated by ET-1 to result in simultaneous sensations of itch and pain, consistent with observations that ET-1 elicits both itch- and pain-related behaviors in animals and burning itch sensations in humans.

Recent studies support roles for neurokinin-1 (NK-1) and gastrin-releasing peptide (GRP) receptor-expressing spinal neurons in itch. We presently investigated expression of substance P (SP) and GRP in pruritogen-responsive primary sensory neurons and roles for these neuropeptides in itch signaling. Responses of dorsal root ganglion (DRG) cells to various pruritogens were observed by calcium imaging. DRG cells were then processed for SP, GRP, and isolectin B-4 (IB4; a marker for nonpeptidergic neurons) immunofluorescence. Of pruritogen-responsive DRG cells, 11.8–26.8%, 21.8–40.0%, and 21.4–26.8% were immunopositive for SP, GRP, and IB4, respectively. In behavioral studies, both systemic and intrathecal administration of a NK-1 receptor antagonist significantly attenuated scratching evoked by chloroquine and a protease-activated receptor 2 agonist, SLIGRL, but not histamine, bovine adrenal medulla peptide 8-22 (BAM8-22), or serotonin. Systemic or intrathecal administration of a GRP receptor antagonist attenuated scratching evoked by chloroquine and SLIGRL but not BAM8-22 or histamine. The GRP receptor antagonist enhanced scratching evoked by serotonin. These results indicate that SP and GRP expressed in primary sensory neurons are partially involved as neurotransmitters in histamine-independent itch signaling from the skin to the spinal cord.

Chronic itch is symptomatic of many skin conditions and systemic diseases. Little is known about pathophysiological alterations in itch-signaling neural pathways associated with chronic itch. We used a mouse model of hindpaw chronic dry skin itch to investigate properties of presumptive itch-signaling neurons. Neurons in the lumbar superficial dorsal horn ipsilateral to hindpaw dry skin treatment exhibited a high level of spontaneous activity that was inhibited by scratching the plantar surface. Most spontaneously active units exhibited further increases in firing rate following intradermal injection of an agonist of the protease-activated receptor PAR-2, or histamine. The large majority of pruritogen-responsive units also responded to capsaicin and allyl isothiocyanate. For neurons ipsilateral to dry skin treatment, responses elicited by the PAR-2 agonist, but not histamine or mechanical stimuli, were significantly larger compared with neurons ipsilateral to vehicle (water) treatment or neurons recorded in naïve (untreated) mice. The spontaneous activity may signal ongoing itch, while enhanced PAR-2 agonist-evoked responses may underlie hyperknesis (enhanced itch), both of which are symptomatic of many chronic itch conditions. The enhancement of neuronal responses evoked by the PAR-2 agonist, but not by histamine or mechanical stimuli, implies that the dry skin condition selectively sensitized PAR-2 agonist-sensitive primary afferent pruriceptors.

Recent studies have suggested the existence of separate transduction mechanisms and sensory pathways for histamine and nonhistaminergic types of itch. We studied whether histamine and an agonist of the protease-activated receptor (PAR)-2, associated with nonhistaminergic itch, excite murine dorsal horn neurons. Single units were recorded in superficial lumbar dorsal horn of adult ICR mice anesthetized with pentobarbital. Unit activity was searched using a small intradermal hindpaw injection of histamine or the PAR-2 agonist SLIGRL-NH2. Isolated units were subsequently challenged with intradermal histamine followed by SLIGRL-NH2 (each 50 μg/1 μl) or reverse order, followed by mechanical, thermal, and algogenic stimuli. Forty-three units were classified as wide dynamic range (62%), nociceptive specific (22%), or mechano insensitive (16%). Twenty units gave prolonged (mean, 10 min) discharges to intradermal injection of histamine; 76% responded to subsequent SLIGRL-NH2, often more briefly. Units additionally responded to noxious heat (63%), cooling (43%), topical mustard oil (53%), and intradermal capsaicin (67%). Twenty-two other units gave prolonged (mean, 5 min) responses to initial intradermal injection of SLIGRL-NH2; 85% responded to subsequent intradermal histamine. They also responded to noxious heat (75%), mustard oil (93%), capsaicin (63%), and one to cooling. Most superficial dorsal horn neurons were excited by both histamine and the PAR-2 agonist, suggesting overlapping pathways for histamine- and non–histamine-mediated itch. Because the large majority of pruritogen-responsive neurons also responded to noxious stimuli, itch may be signaled at least partly by a population code.