Morphological And Functional Investigations On The Spinal-Parabrachial-Midline/Intralaminar Thalamic Nucleus Pathway Involved In Transmission And Modulation On The Itch Sensation

Itch is an annoying sensation that triggers a desire to scratch.The acute itch sensation,like a mosquito bite,can help people to avoid environmental noxious stimuli;whereas chronic itch dramatically interferes with life quality of patients.To date,although numerous researches have been made on the peripheral mechanism of itch sensation,how does itch signal been transmitted to the higher center on the supraspinal level remains elusive.Due to the unclarified mechanism of itch and pain,itch and pain sensation were once regarded as the same,and itch is merely weak version of pain.In recent years,it has been proved that gastrin releasing peptide receptor(GRPR)-expressing excitatory interneurons in spinal dorsal horn(SDH)specifically send itch signals to the parabrachial nucleus(PBN)without interfering with pain sensation.This research suggests that the "labeled line" theory might be a candidate explanation of itch transmission.Meanwhile,PBN is one of the essential transit nodes of sensory information in pons that receives neural terminals from the substance P receptor(SPR)-expressing neurons in SDH.It transmits noxious information further to the thalamus,amygdala,hypothalamus and periaqueductal grey matter Prior researches demonstrat that the number of FOS-immunoreactive(-ir)neurons increase significantly in the dorsal midline and intralaminar complex(dMITC)in acute itch model,suggesting its participation in itch sensation.Morphological evidence also shows that dMITC receives abundant projections from the PBN,and has emphasized the paraventricular thalamic nucleus(PVT)and central medial thalamic nucleus(CM)as main target nuclei of itch-activated neurons in the PBN.Based on the evidence above,whether there exists a direct pathway from the SDH to the dMITC that participates in itch sensation remains unknown.Besides,do PVT and CM receive PBN projections concerning itch sensation,and if so,do these modulatory effects require subpopulation specificity?What is the nest key node of PBN-PVT/CM projections in the limbic system?In the present study,morphological,optogenetic,pharmacological,fiber photometry,designer receptor exclusively activated by designer drugs(DREADD)technologies and electrophysiological technologies were utilized.We attempted to provide morphological evidence of a possible direct and/or indirect pathway from the SDH to the dMITC.Furthermore,PBN-PVT/CM neuronal pathway was modified to validate their roles in itch or pain sensation,and in anxiety-like behavior.The contents of the present work are divided into five parts:1.Morphological investigations on the the direct and indirect pathway from the SDH to the dMITC(1)The direct SDH-dMITC/PBN neuronal pathway in mice:The retrograde tracer Fluorogold(FG)was injected into the PBN or dMITC,respectively,and performed with acute itch model.Combined with immunofluorescence staining of the coronal sections of spinal cord,the SPR/FOS/FG triple-labeled neurons were observed on the superficial laminae of the SDH.(2)Collateral projections from the SDH to the PBN and dMITC:Mice were stereotactically injected with tetramethylrhodamine-dextran(TMR)into the left dMITC and FG into the PBN,respectively,and performed with acute itch model.TMR/FG double-labeled collateral projection neurons were observed mainly in superficial SDH,and some of them were SPR-immunoreactive(-ir)and were activated in His-induced acute itch model.(3)The indirect SDH-PBN-dMITC pathway in mice:anterograde viral vector and FG were used to delineate indirect pathway.Some dMITC-projecting neurons in the PBN were activated by itch stimulation,and were in close contact with terminals projecting form the SDH.Meanwhile,viral strategy was used to determine an indirect pathway.Abundant fibers and terminals were observed in multiple subnuclei of the dMITC.These results indicated the direct SDH-dMITC pathway,collateral projections from SDH to the PBN and dMITC,and indirect SDH-PBN-dMITC pathway in mice central nervous system,which were involved in itch signal transmission.2.Morphological and functional investigations on the participation of the PVT in itch sensation(1)Activation of the PVT with acute itch stimuli:acute pruritogens were intradermally injected into the nape of mice to induce acute itch models.Significant more FOSimmunoreactive(FOS-ir)neurons were observed in PVT.Behaviorally,the scratching behavior and paw withdraw threshold(PWT)decreased after PVT activation,indicating a modulatory role of the PVT in pruriception.(2)PBN-PVT pathway in itch sensation:Retrograde tracer FG was injected into the PVT,and most FG-labeled neurons in PBN were colocalized with vesicular glutamate transporter 2(VGluT2)signal.Some of the VGluT2/FG double-labeled neurons were FOS-ir,indicating that the PBN-PVT glutamatergic pathway was activated by acute itch stimulation.(3)Modulatory roles of the PBN-PVT pathway in itch:Chemogenetic viral strategy was used to specifically modify the PBN-PVT pathway.Activation of PBN-PVT pathway significantly alleviated the scratching behavior whereas reduced PWT.Meanwhile,inhibiting the same pathway increased spontaneous scratching without affecting PWT.Furthermore,optogenetic activation of the PBN-PVT pathway significantly attenuated scratching behavior,which also induced allodynia and anxiety-like behavior.(4)PVT AMPA receptor-expressing neurons in itch signal modulation:To identify the neurochemical properties of the PVT neurons in itch sensation,optogenetic and pharmacological technics were utilized.Optogenetic activation of the PBN-PVT pathway resulted in attenuated scratching behavior and decreased PWT.Furthermore,when PVT was applied with 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX),an AMPA receptor antagonist,the spontaneous scratching behavior of mice increased,which cannot be suppressed by optogenetic activation.These results suggested that PVT received glutamatergic projections from PBN,and the PBN-PVT pathway participated in itch modulation through AMPA receptor-expressing neurons in PVT.Furthermore,activation of the PBN-PVT pathway attenuated scratching behavior,which also induced allodynia and anxiety-like behavior.3.Modulatory role of the target nuclei of the PVT in itch,pain and anxiety-like behavior(1)PVT projections to the prelimbic cortex(PrL),central nucleus of the amygdala(CeA)and insula cortex(IC):The anterograde virus AAV-CMV-bGlobin-Cre-mCherry was injected into the PBN with AAV-hSyn-DIO-eGFP injected into the PVT.The eGFPlabeled fibers were observed in PrL,CeA and IC.Retrograde tracer FG was injected into these nuclei in three groups of mice to delineate the rostral-caudal distribution of three subpopulations of neurons.Afterwards,three different retrograde tracers were injected into the PrL,CeA and IC,respectively.The percentage of collateral projection to CeA and IC is significantly higher.These results provided morphological foundations for the diverse modulatory roles of the PBN-PVT pathway in future behavioral assays.(2)The involvement of PBN-PVT-PrL/CeA/IC pathway in anxiety-like behavior,pruriception and nociception:to further clarify the specific modulatory roles of three target nuclei,viral strategy was used to specifically knock-down different pathways.Meanwhile,photoactivation of the PBN-PVT pathway was performed.It was observed that PBN-PVT-CeA/IC pathways were involved in itch and pain modulation;whereas the PBN-PVT-PrL pathway played a modulatory role in anxiety-like behavior.(3)The electrophysiological characteristics of PrL/CeA/IC-projecting neurons in PVT:It was found that the excitabilities of PrL-projecting PVT neurons(PVTPrL)decreased dramatically,whereas the excitabilities of PVTIC and PVTCeA neurons both increased with chronic itch and pain stimuli.Meanwhile,in SNI and DNFB models,the connectivity of PBN and PVTCeA neurons were strengthened,whereas the connectivity of PBN and PVTIC neurons were more enhanced in SNI models.Consequently,the PBN-PVT pathway participated in pain,itch and negative emotions.The PBN-PVT-PrL pathway was involved in anxiety-like behavior;whereas the PBN-PVTCeA/IC pathways exhibited modulatory effects in both pruriception and nociception.4.Morphological and functional investigations on the central medial thalamic nucleus(CM)in itch transmission(1)Participation of CM in itch transmission:both acute and chronic itch models were effective to induce an increase of FOS-ir neurons in the CM.Afterwards,fiber photometry was performed,and the population calcium signal increased significantly at the onset of scratching behavior.In electrolytic lesion of CM,the scratching behavior in both acute and chronic itch models were relieved in lesion group,with an attenuated trend in anxiety-like behavior induced by DNFB model.(2)Pruriceptive projections from PBN to CM:Abundant eYFP-labeled fibers and terminals were observed in CM when the activity-dependent virus was injected into the PBN with DNFB model performed.Besides,the retrograde tracing of FG combined with fluorescence in situ hybridization(FISH)technology demonstrated that about 80%of the itch-activated,CM-projecting neurons are VGluT2+neurons.By specifically modify the population calcium signal of the PBN-CM pathway,it was found that the calcium signal increased align with scratching behavior.Furthermore,optogenetic silence of the PBN-CM pathway exhibited an inhibitory effect of scratching behavior.(3)The PBN-CM-mPFC pathway in itch signal transmission and itch-related anxiety-like behavior:CM neurons receiving PBN projection sent its fibers to the mPFC.Furthermore,the chemogenetic activation of the CM-mPFC pathway exhibited no specific behavioral changes,whereas inhibiting this pathway attenuated both acute and chronic itch-induced scratching behavior,and alleviated anxiety-like behavior induced by chronic itch model.These results validated the involvement of the PBN-CM-mPFC pathway in itch signal transmission,and suppressing the CM-mPFC pathway attenuated the scratching behavior and chronic itch-induced anxiety-like behavior,simultaneously.5.The dynamic participation of the CM-mPFC pathway in acute and chronic itch models.(1)The electrophysiological characteristics of mPFC-projecting CM neurons(CMmPFC)in acute and chronic itch models:the CMmPFC neurons were specifically labeled with retrograde tracer with AAV-CaMKII-ChR2-mCherry injected into the PBN.The excitatory postsynaptic transmission of CM neurons in DNFB model elevated significantly.It was also observed that the intrinsic properties of CMmPFC neurons increased with both acute and chronic itch stimuli,and the CMmPFc neurons in DNFB group exhibited higher neuronal excitability.(2)The strengthened CM-mPFCINs in chronic itch model:the AAV-hSyn-eGFPSynaptophysin-mRuby was injected into the CM,which exhibited CM connections with both pyramidal neurons(PNs)and interneurons(INs)in mPFC.Functionally,AAVCaMKII-ChR2-mCherry was injected into the CM,and mPFC was activated through an objective lens.The number of spiking exhibited an increasing trend in acute itch model in optogenetic activation,which decreased significantly in chronic itch model.(3)The CM-related mPFC excitatory/inhibitory imbalance in DNFB model:CM sent monosynaptic projections to both PNsmPFC and INsmPFC,which also induced the feedforward inhibition of the PNsmPFC.Furthermore,the paired-pause ratio(PPR)results exhibited an increased presynaptic releasing probability of the PNsmPFC in His model.A decreased PPR of INsmPFC were observed in DNFB model,along with the increased NMDA current of PNsmPFC with chronic itch stimulation.Finally,Lanicemine,a NMDA antagonist attenuated the scratching behavior and chronic itch-induced anxiety-like behavior,simultaneously.All above-mentioned results proved a dynamic participation of the CM-mPFC pathway in acute and chronic itch models.Meanwhile,the electrophysiological results demonstrated a strengthened feedforward inhibition of PNsmPFC in DNFB model(E/I imbalance).Lanicemine,the NMDA antagonist,could effectively attenuate both the scratching behavior and anxiety-like behavior induced by chronic itch model.Conclusions:The present results have,at least partially,clarified the itch sensory signal transmission pathway on the supraspinal level.It has demonstrated that:①SDH projecting neurons send itch sensory signal to the dMITC through SDHdMITC direct pathway,SDH-dMITC/PBN collateral projections and SDH-PBN-dMITC indirect pathway.②PVT receives itch sensory information through glutamatergic projections from the PBN,and transmits it further to PrL,CeA and IC,which play modulatory roles in negative emotions,pruriceptive and nociceptive sensation,respectively.③PBN-CM-mPFC pathway is involved in itch sensory signal transmission.The ascending projection from CM to mPFC processes enhanced connections with PNsmPFC in acute itch model,whereas the same projection processes enhanced connections with INsmPFC with chronic itch stimuli.