Optotagging and characterization of GABAergic rostral ventromedial medulla (RVM) neurons.

The transmission of nociceptive and pruriceptive signals in the spinal cord is greatly influenced by descending modulation from brain areas such as the rostral ventromedial medulla (RVM). Within the RVM three classes of neurons have been discovered which are relevant to spinal pain modulation, the On, Off, and Neutral cells. These neurons were discovered due to their functional response to nociceptive stimulation. On cells are excited, Off cells are inhibited, and Neutral cells have no response to noxious stimulation. Since these neurons are identified by functional response characteristics it has been difficult to molecularly identify them. In the present study, we leverage our ability to perform optotagging within the RVM to determine whether RVM On, Off, and Neutral cells are GABAergic. We found that 27.27% of RVM On cells, 47.37% of RVM Off cells, and 42.6% of RVM Neutral cells were GABAergic. These results demonstrate that RVM On, Off, and Neutral cells represent a heterogeneous population of neurons and provide a reliable technique for the molecular identification of these neurons

Transmitters and Pathways Mediating Inhibition of Spinal Itch-Signaling Neurons by Scratching and Other Counterstimuli

Scratching relieves itch, but the underlying neural mechanisms are poorly understood. We presently investigated a role for the inhibitory neurotransmitters GABA and glycine in scratch-evoked inhibition of spinal itch-signaling neurons in a mouse model of chronic dry skin itch. Superficial dorsal horn neurons ipsilateral to hindpaw dry skin treatment exhibited a high level of spontaneous firing that was significantly attenuated by cutaneous scratching, pinch and noxious heat. Scratch-evoked inhibition was nearly abolished by spinal delivery of the glycine antagonist, strychnine, and was markedly attenuated by respective GABAA and GABAB antagonists bicuculline and saclofen. Scratch-evoked inhibition was also significantly attenuated (but not abolished) by interruption of the upper cervical spinal cord, indicating the involvement of both segmental and suprasegmental circuits that engage glycine- and GABA-mediated inhibition of spinal itch-signaling neurons by noxious counterstimuli

Spontaneous itch in the absence of hyperalgesia in a mouse hindpaw dry skin model

We presently investigated if chronic dry skin treatment of the mouse hindpaw results in itch-related behavior, with or without accompanying hyperalgesia and allodynia. Following a 10-day period of treatment of one hindpaw with a mixture of acetone-diethylether-water (AEW), mice exhibited a significant increase in spontaneous biting behavior directed to the treated hindpaw compared to control animals in which one hindpaw was similarly treated with water only (W). Biting in the AEW group was significantly attenuated by the µ-opioid antagonist naltrexone but was unaffected by the µ-agonist morphine. There were no significant differences in hindpaw heat withdrawal latency, mechanical withdrawal threshold, or cold-plate latency between W and AEW treatment groups. These results indicate that chronic AEW treatment induces spontaneous itch but does not alter pain sensitivity, supporting the utility of this model for studies of chronic dry skin itch

Role of thermosensitive transient receptor potential (TRP) channels in thermal preference of male and female mice

Transient Receptor Potential (TRP) ion channels are important for sensing environmental temperature. In rodents, TRPV4 senses warmth (25-34 °C), TRPV1 senses heat (>42 °C), TRPA1 putatively senses cold (<17 °C), and TRPM8 senses cool-cold (18-26 °C). We investigated if knockout (KO) mice lacking these TRP channels exhibited changes in thermal preference. Thermal preference was tested using a dual hot-cold plate with one thermoelectric surface set at 30 °C and the adjacent surface at a temperature of 15-45 °C in 5 °C increments. Blinded observers counted the number of times mice crossed through an opening between plates and the percentage of time spent on the 30 °C plate. In a separate experiment, observers blinded as to genotype also assessed the temperature at the location on a thermal gradient (1.83 m, 4-50 °C) occupied by the mouse at 5- or 10-min intervals over 2 h. Male and female wildtype mice preferred 30 °C and significantly avoided colder (15-20 °C) and hotter (40-45 °C) temperatures. Male TRPV1KOs and TRPA1KOs, and TRPV4KOs of both sexes, were similar, while female WTs, TRPV1KOs, TRPA1KOs and TRPM8KOs did not show significant thermal preferences across the temperature range. Male and female TRPM8KOs did not significantly avoid the coldest temperatures. Male mice (except for TRPM8KOs) exhibited significantly fewer plate crossings at hot and cold temperatures and more crossings at thermoneutral temperatures, while females exhibited a similar but non-significant trend. Occupancy temperatures along the thermal gradient exhibited a broad distribution that shrank somewhat over time. Mean occupancy temperatures (recorded at 90-120 min) were significantly higher for females (30-34 °C) compared to males (26-27 °C) of all genotypes, except for TRPA1KOs which exhibited no sex difference. The results indicate (1) sex differences with females (except TRPA1KOs) preferring warmer temperatures, (2) reduced thermosensitivity in female TRPV1KOs, and (3) reduced sensitivity to cold and innocuous warmth in male and female TRPM8KOs consistent with previous studies

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

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-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 id 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

Targeting Transient Receptor Potential (TRP) Channels, Mas-Related G-Protein-Coupled Receptors (Mrgprs), and Protease-Activated Receptors (PARs) to Relieve Itch.

Itch (pruritus) is a sensation in the skin that provokes the desire to scratch. The sensation of itch is mediated through a subclass of primary afferent sensory neurons, termed pruriceptors, which express molecular receptors that are activated by itch-evoking ligands. Also expressed in pruriceptors are several types of Transient Receptor Potential (TRP) channels. TRP channels are a diverse class of cation channels that are responsive to various somatosensory stimuli like touch, pain, itch, and temperature. In pruriceptors, TRP channels can be activated through intracellular signaling cascades initiated by pruritogen receptors and underly neuronal activation. In this review, we discuss the role of TRP channels TRPA1, TRPV1, TRPV2, TRPV3, TRPV4, TRPM8, and TRPC3/4 in acute and chronic pruritus. Since these channels often mediate itch in association with pruritogen receptors, we also discuss Mas-related G-protein-coupled receptors (Mrgprs) and protease-activated receptors (PARs). Additionally, we cover the exciting therapeutic targets amongst the TRP family, as well as Mrgprs and PARs for the treatment of pruritus