The Characterization Of Peripheral Distribution And Electrophysiology Of Itch-specific MrgprA3~+ Neuron

Itch is defined as an unpleasant or irritating skin sensation that elicits the desire or reflex to scratch. The exogenous or endogenous pruritic agents activate the receptor on the peripheral fibers of pruriceptors, and then the pruriceptors are activated and generate the action potentials(APs), which are conducted to the interneuron in spinal dorsal horn via its central fibers. The pruritic signal is sent to the sensory cortex via pruriceptive spino-thalamic tract neurons. Finally, the itch sensation is formed in the brain. MrgprA3 is a member of Mrgprs family, which is specifically expressed in the dorsal root ganglion(DRG) and trigeminal ganglion(TG). MrgprA3 functions as a receptor for chloroquine(CQ), which is used as a drug to treat malaria. MrgprA3-expressing neurons(MrgprA3~+ neurons) are a subpopulation of nociceptors specifically linked to itch, which was first discovered in the peripheral nervous system in mice. Generally, CQ activates MrgprA3 and then excites the MrgprA3~+ neuron, and finally induces an itch sensation in mice. In the present study, we characterized the peripheral distribution and electrophysiology of itch-specific MrgprA3~+ neuron.We first labeled the MrgprA3~+neuron using a genetic method. Generally, we crossed the transgenic Mrgpra3EGFP-Cre mice with Cre-dependent ROSA26 td Tomato mice,and obtained the transgenic Mrgpra3EGFP-Cre;ROSA26td Tomato offerspring mice, in which the MrgprA3~+ neurons in the DRG tissues were marked by the coexpression of td Tomato and EGFP protein. It provided an important tool for our current study. We characterized the projection distribution of peripheral MrgprA3~+ fibers in the skin tissues by using confocal imaging technology. The results indicated that the MrgprA3~+fibers in the neck and abdomen skin were sparse with low density and showed scattered dotted feature. The MrgprA3~+fibers in the back skin were slenderly distributed. In this skin region, some hair follicles were twined by MrgprA3~+ fibers. By contrast, the MrgprA3~+ fibers located in the cheek and paw skin were redundantly distributed with higher density. The diameter of MrgprA3~+ fibers was also significantly greater compared with other skin regions.Then, we characterized the three dimensional distribution of cell body of MrgprA3~+ neurons in the intact DRG tissues, which were located in different spinal level. Our results suggested that the Z axis depth for cervical DRG was 350 μm. The all MrgprA3~+neurons in it were small-diameter sensory neurons. The diameter is mainly in11~20 μm interval with the ratio of 90.7%. The Z axis depth for thoracic DRG was 250μm. The majority of MrgprA3~+neurons were small-diameter sensory neurons. The diameter is also mainly in 11~20 μm interval with the ratio of 81.1%. In addition, 1.0%MrgprA3~+ neurons in the thoracic DRG were medium-diameter sensory neurons with diameter over 26 μm. The Z axis depth for lumbar DRG was 400 μm. All of MrgprA3~+neurons were small-diameter sensory neurons. The cell body of MrgprA3~+neurons showed clear morphology and were intensely distributed. The diameter was mainly in16~25 μm interval with the ratio of 85.6%. However, the Z axis depth for sacral DRG was only 200 μm. The MrgprA3~+ neurons were sparsely distributed with low density, all of them belonged to small-diameter sensory neurons. The diameter was mainly in 11~20 μm interval with the ratio of 92.3%. Surprisingly, we could obviously observe the MrgprA3~+ fibers in the sacral DRG, which were labeled by td Tomato with red fluorescence but not EGFP with green fluorescence. Thus, MrgprA3~+ neurons were existed in the four spinal DRG tissues(cervical, thoracic, lumbar and sacral DRG).However, the amounts and features of MrgprA3~+neurons were significantly different among these DRG tissues.Lastly, we comparably investigated the electrophysiological property between MrgprA3~+ neurons and two subsets of MrgprA3- neurons(MrgprA3--non-Dil neurons and MrgprA3--Dil neurons) by conducting patch-clamp recording. MrgprA3--Dil neurons were a subpopulation of MrgprA3- neurons specifically innervating skin, which were labeled with retrograde tracing method by Dil dye. Thus, MrgprA3--Dil neurons had similar innervating features with MrgprA3~+ neurons. But MrgprA3--non-Dil neurons have more innervating regions, since they might innervate the muscle, bone and other visceral tissue, in addition to peripheral skin tissues. Our results indicated that MrgprA3~+ neurons showed single action potential(AP) firing pattern in the most of cases, however, multiple APs firing pattern was exsited in two subsets of MrgprA3-neurons. By analyzing the their AP-related parameters, we found that both duration to50% and duration to 80% decay, two parameters in the AP afterdepolarization phase,were significantly greater in the MrgprA3~+ neurons compared with two subsets of MrgprA3- neurons. This suggested that the membrane potential of MrgprA3~+ neurons needed more time to recover the resting membrane potential(RMP) level after an AP generation. Thus, the neuronal excitability of MrgprA3~+ neurons is significantly higher than two subsets of MrgprA3- neurons. We next comparably studied their voltage-gated potassium(Kv) channels by using whole cell voltage-clamp recording. The results indicated that MrgprA3~+neurons had significantly higher sustained Kv currents compared with two subsets of MrgprA3- neurons. Strikingly, the RMP of MrgprA3~+neurons significantly depolarized when blocking sustained Kv channels with TEA.Importantly, blocking sustained Kv channels significantly enhanced the MrgprA3~+neurons’ AP firing activity, which indicated that sustained Kv channels reduced the MrgprA3~+ neurons’ excitability. Besides, we also comparably studied their voltage-gated sodium(Nav) channels. The results showed that the TTX-R Nav peak currents density was significantly greater in the MrgprA3~+ neurons in relative to two subsets of MrgprA3-neurons. However, there were no significant difference in steady state activation and inactivation between MrgprA3~+ neurons and two subsets ofMrgprA3-neurons. Since the AP firing activity was significantly weaker in the MrgprA3~+ neurons compared with two subsets of MrgprA3- neurons, the MrgprA3~+neurons’ excitability was predominantly modulated by sustained Kv channels. We speculated that TTX-R Nav channels in MrgprA3~+neurons had a balanced role to sustained Kv channels, avoiding its neuronal excitability was further reduced. This might be essential for the maintenance of MrgprA3~+ neurons’ normal physiological function.In summary, we first characterized the distribution of MrgprA3~+fibers in the skin tissue and the three dimensional distribution of the cell body of MrgprA3~+ neurons in the intact DRG tissue. Furthermore, we also first investigated the electrophysiological properites of MrgprA3~+neurons and elucidated the mechanism of ion channels. Taken together, the present study could contribute to further understand the morphological features and physiological functions of itch-specific neurons. It also might provide instructions for developing anti-pruritic drugs and itch disease therapy.