Morphological And Functional Studies On Serotoninergic Ascending Projection From Dorsal Raphe Nucleus To Insular Cortex Is Involved In Acute Itch Sensation Processing In Mice

Background & Objectives: Itch is an unpleasant feeling that causes the urge and behavior of scratching,which can be caused by a variety of local,systemic,psychiatric,and neurological diseases,or by different sources of itching.The mechanism of itch has been revealed in a certain degree in the peripheral sensory,spinal nerve and spinal cord levels,however the mechanism of itch in the central neural system,especially the cerebral cortex,is mainly limited to imaging studies now.The itch-activated cerebral cortex is not a specific brain region,rather than a complex matrix network.For the conductive pathway of itch information,there is still no study of the pathway above the spinal cord level and its cellular and molecular mechanisms,but the itch sensory center is observed by imaging techniques,showing that the thalamic(thalamic nucleus),cerebral cortex like anterior cingular cortex(ACC),insular cortex(IC),and medial prefrontal cortex(m PFC)etc,are associated with itch susceptibility.The peripheral itching mechanism of serotonin(5-HT)is dependent on histamine receptors and is associated with enhanced itch.5-HTergic neurons from the dorsal raphe nucleus(DRN)are important components of the central endogenous pain regulation system and are also involved in the regulation of various physiological functions of the body.The descending 5-HT system acts on the neurons which express both 5-HT1 A receptor and gastrin-releasing peptide receptor(GRPR)in the spinal dorsal horn(SDR),facilitating the transmission of itch.It can be seen that 5-HT plays an important role in itch transmission and regulation,while DRN contains a mass of the 5-HTergic neurons,some of which send ascending closed fibers connected to the high cerebral cortex including IC.The 5-HT ascending projections might be another new pathway for itch signaling and regulation,which provides a new direction for revealing the mechanism and clinical treatment of chronic pruritus.Methods: Firstly,we performed immunohistochemical staining to map the areas(including DRN and IC)in with FOS protein and p-ERK were evoked by histamine (His)and chloroquine(CQ)-induced acute itch.Secondly,using antegrade and retrograde tract tracing techniques,immunohistochemical multiple staining to observe whether there exists of DRN-IC pathway and whether 5-HTergic ascending projection is involved in itch transmission.Thirdly,pharmacogenetic approach excite DRN-IC pathway,were applied to observed scratching behaviors changes in normal and itch model animals.Results:1.Compared with the sham group(Saline group),animals with His and CQ intradermal injection showed obvious scratching,both FOS-and p-ERK-immunoreactive neurons were increased in the DRN and IC in the acute itch mice.Acute itch activated part of the neurons in DRN and IC.2.After injecting the antegrade tracer(biotin dextran amine,BDA)into the DRN,it was observed that there were quite dense BDA-labeled a "bead-like" fibers and terminals in the IC;After a retrograde tracer(fluorescent gold,FG)was injected into the IC and combined with immunofluorescence double-labeled staining,it was observed that almost all FG-labeled neuronal cell bodies in the DRN showed 5-HT immunoreactive positive staining.3.The results of immunofluorescence triple staining indicated that acute itch stimuli induced FOS expression in 5-HTergic DRN-IC projecting neurons within the DRN.FG/5-HT/ FOS triple-labeled neurons were scattered in the DRN.4.Chemogenetically activating the DRN-IC pathway did not affect the spontaneous itching behavior,but reduced scratch numbers in both histamine and chloroquine acute itch(P < 0.05).Conclusions: Our present findings suggest that both the IC and the DR are involved in the sensory processing of acute itch,which is consistent with imaging data.In addition,5-HT-ergic neurons in the DR send projections toward the IC,and this ascending pathway is activated under acute itch status and might play important roles in the transmission of itch sensory information.Chemogenetically activating this pathway inhibits the scratching behavior of acute itch mice.The results of the present our study might provide new pathway and morphological evidence for the transmission and regulation of itch information in the central nervous system.