Mechanism Of ER_?⁺ Neurons In Spinal Dorsal Horn Promoting Itch Sensation

Objective:Observe whether there is gender difference in itching behavior of mice,and the role of estrogen in the gender difference of itch sensation was observed.Further study on the mechanism of estrogen receptor alpha(ER?)in spinal dorsal horn to promote pruritus,providing new experimental basis and treatment ideas for estrogen-related pruritus.Methods:Firstly,the scratching behavior(itch behavior)of male and female C57mice after intradermal injection of CQ/His was observed through itch behavior test,and the exogenous effect of estrogen level change on itch sensation in male and female mice was induced.And the effects of specific agonists or antagonists of ER?,ER?and G protein coupled estrogen receptor(GPER)on itch behavior induced by CQ.Secondly,the effect of activating or inhibiting ER?in spinal dorsal horn on CQ induced itching behavior was observed by chemical genetic method.Thirdly,the relationship between ER?and gastrin releasing peptide(GRP),which is closely related to CQ itching,was observed in the spinal dorsal horn by immunofluorescence histochemical staining and immunoelectron microscopy.Finally,whole-cell patch clamp recording technique was used to observe the effect of GRP on excitability of ER?⁺neurons in the superficial dorsal horn of spinal cord of mice.Results:(1)Compared with C57 male mice,the number of scratching induced by intracutaneous injection of CQ/His in the back of the neck of female mice was significantly higher than that of male mice,indicating that there were gender differences in itch sensitivity.(2)The scratching behavior induced by CQ was increased by subcutaneous injection of estrogen into the abdomen of C57 male mice.However,with the decrease of estrogen level in ovarioectomized female mice,the CQ-induced scratching behavior also decreased significantly.(3)Intrathecal injection of ER?specific agonist PPT could significantly increase the scratching behavior induced by CQ,while intrathecal injection of ER?specific antagonist AZD9496 can reverse the promoting effect of estrogen on CQ induced pruritus.Intrathecal administration of ER?specific agonist DPN and antagonist PHTPP,GPER specific agonist G1 and antagonist G15 did not change the itching behavior induced by CQ.(4)The scratching behavior induced by CQ was increased or decreased in ER?-Cre transgenic mice by injection of ER?activated or inhibited chemogenetic virus into the spinal dorsal horn.(5)The GRP immunofluorescence staining of ER?-td Tomato double labeled mice showed that the GRP positive fiber terminals in the superficial dorsal horn of spinal cord were in close contact with ER?⁺neurons,and the immunoelectron microscopy showed that the GRP positive axon terminals formed asymmetric(excitatory)synaptic connections with the dendrites of ER?⁺neurons.(6)The results of whole-cell patch clamp showed that the amplitude of spontaneous excitatory postsynaptic current(EPSC)in spinal dorsal horn ER?⁺neurons increased significantly under the action of GRP,while the excitability of ER?⁺neurons decreased significantly under the action of GRP receptor(GRPR)antagonist.Conclusion:The itching sensitivity of normal female mice to CQ was significantly higher than that of male mice,which was mainly mediated by estrogen(acting on ER?).Exogenous injection of estrogen can increase CQ induced acute itching behavior,which can be simulated by ER?agonists.In the superficial layer of spinal dorsal horn,estrogen and GRP may act on ER?neurons at the same time,and promote itching through the synergistic effect of ER?and GRPR.