E. Carstens

Activation of Superficial Dorsal Horn Neurons in the Mouse by a PAR-2 Agonist and 5-HT: Potential Role in Itch

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 (id) injection of the PAR-2 agonist SLIGRL-NH2 in the rostral back evoked bouts of directed hindlimb scratches over 20–30 min. Hindpaw injection of SLIGRL-NH2 produced Fos staining in superficial dorsal horn which was then targeted for single-unit recording. Small id microinjections of SLIGRL-NH2 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 id SLIGRL-NH2 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-NH2, mustard oil, and capsaicin. The prolonged responses of superficial dorsal horn neurons to SLIGRL-NH2 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.

Facial Injections of Pruritogens and Algogens Excite Partly Overlapping Populations of Primary and Second-Order Trigeminal Neurons in Mice

Intradermal cheek injection of pruitogens or algogens differentially elicits hindlimb scratching or forelimb wiping, suggesting that these behaviors distinguish between itch and pain. We studied whether pruritogens and algogens excite separate or overlapping populations of primary afferent and second-order trigeminal neurons in mice. Calcium imaging of primary sensory trigeminal ganglion (TG) cells showed that 15.4% responded to histamine, 5.8% to the protease-activated receptor (PAR)-2 agonist, 13.4% to allyl isothiocyanate (AITC), and 36.7% to capsaicin. AITC and/or capsaicin activated the vast majority of histamine- and PAR-2 agonist-sensitive TG cells. A chemical search strategy identified second-order neurons in trigeminal subnucleus caudalis (Vc) responsive to histamine, the PAR-2 agonist, or AITC. A minority of histamine or PAR-2 agonist-responsive Vc neurons responded to the other pruritogen, whereas a large majority of puritogen-responsive Vc neurons responded to capsaicin and/or AITC. A minority of AITC-responsive Vc neurons responded to pruritogens, whereas most responded to capsaicin. These data indicate that most primary and higher-order trigeminal sensory neurons are activated by both pruritic and algesic stimuli, although a minority exhibit selectivity. The results are discussed in terms of population codes for itch and pain that result in distinct behavioral responses of hindlimb scratching and forelimb wiping that are mediated at lumbar and cervical segmental levels, respectively.

Excitation of Mouse Superficial Dorsal Horn Neurons by Histamine and/or PAR-2 Agonist: Potential Role in Itch

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 microg/1 microl) 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.

Enhanced responses of lumbar superficial dorsal horn neurons to intradermal PAR-2 agonist but not histamine in a mouse hindpaw dry skin itch model

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.

Opposing effects of cervical spinal cold block on spinal itch and pain transmission

Inactivation of descending pathways enhanced responses of spinal dorsal horn neurons to noxious stimuli, but little is known regarding tonic descending modulation of spinal itch transmission. To study effects of cervical spinal cold block on responses of dorsal horn neurons to itch-evoking and pain-evoking stimuli, single-unit recordings were made from superficial dorsal horn wide dynamic range and nociceptive-specific-type neurons in pentobarbital-anesthetized mice. Intradermal histamine excited 17 units. Cold block stating 1 minute after intradermal injection of histamine caused a marked decrease in firing. The histamine-evoked response during and following cold block was significantly lower compared with control histamine-evoked responses in the absence of cold block. A similar but weaker depressant effect of cold block was observed for dorsal horn unit responses to chloroquine. Twenty-six units responded to mustard oil allyl isothiocyanate (AITC), with a further significant increase in firing during the 1-minute period of cold block beginning 1 minute after AITC application. Activity during cold block was significantly greater compared with the same time period of control responses to AITC in the absence of cold block. Ten units’ responses to noxious heat were significantly enhanced during cold block, while 6 units’ responses were reduced and 18 unaffected. Cold block had no effect on mechanically evoked responses. These results indicate that spinal chemonociceptive transmission is under tonic descending inhibitory modulation, while spinal pruriceptive transmission is under an opposing, tonic descending facilitatory modulation.

Site- and state-dependent inhibition of pruritogen-responsive spinal neurons by scratching

The relief of itch by scratching is thought to involve inhibition of pruritogen‐responsive neurons in the spinal cord. We recorded the responses of superficial dorsal horn neurons in mice to intradermal injection of the pruritogens chloroquine and histamine. Scratching within an area 5–17u2003mm distant from the injection site, outside of the units’ mechanoreceptive fields (off‐site), significantly inhibited chloroquine‐evoked and histamine‐evoked responses without affecting capsaicin‐evoked firing. This is consistent with observations that scratching at a distance from a site of itch is antipruritic. In contrast, scratching directly at the injection site (within the receptive field; on‐site) had no effect on chloroquine‐evoked neuronal firing, but enhanced the same neurons’ responses to intradermal injection of the algogen capsaicin. Moreover, neuronal responses to histamine were enhanced during on‐site scratching, and this was followed by suppression of firing below baseline levels after termination of scratching. Scratching thus inhibits pruritogen‐responsive neurons in a manner that depends on the input modality (i.e. pain vs. histamine‐dependent or histamine‐independent itch) and skin location.