| As one of the most common and intractable chronic pain,neuropathic pain（NP）is caused by the impairment or disease of the somatosensory nervous system,which is characterized by allodynia,hyperalgesia and spontaneous pain.The pathogenesis of neuropathic pain is complex and ambiguous,NP often accompanied by the change of psychological state,and the influence of social factors,is still a great challenge for basic medicine and clinical medicine.The endocannabinoid system（ECS）has been studied for hundreds of years,which includes ligands such as 2-AG and AEA,receptors CB1 and CB2 and metabolic enzymes such as FAAH.It has been confirmed that endocannabinoids（e CBs）not only have analgesic effect,but also can cause pain in some cases.Studies have confirmed that CB1receptor is also expressed in astrocytes,and a small amount of expression can play an important role.ECS is a key regulator of astrocyte activity.Postsynaptic e CBs can increase intracellular calcium level by activating CB1 receptor on astrocytes,then activate presynaptic NMDAR or m Glu R1,and finally dynamically regulate neuronal excitability and synaptic plasticity.At present,more and more studies suggest that its function is not limited to supporting neurons.People gradually realize that its interaction with neurons plays an important role in many physiological and pathological processes.Astrocytes and neurons are coupled by chemical synapses and electrical synapses.Studies show that there are synaptic connections between pyramidal cells and GABAergic interneurons in hippocampal CA1,and the repetitive discharge between them can activate GABA receptor of nearby astrocytes,increase the intracellular calcium level of astrocytes,induce astrocytes to release glutamate,and finally enhance the inhibitory transmission from GABAergic neurons to pyramidal cells.Our previous study found that there is an inhibitory circuit from glycine（Gly）to PKCγneurons in the spinal cord dorsal horn.In neuropathic pain state,the dysfunction of this circuit makes the tactile information upload to the pain pathway through Aβfiber,which eventually leads to the occurrence of allodynia.There were also a series of changes existed in PKCγneurons,Gly T2 neurons and inhibitory circuits between them.Therefore,we proposed several scientific questions:1.How the endocannabinoids content change after peripheral nerve injury in the spinal cord dorsal horn?2.Do astrocytes express CB1 receptors?Can the change of endocannabinoids content in the spinal cord dorsal horn affect the activation of astrocytes and ultimately alter pain behavior in experimental animals?3.Do changes in the activation state of astrocytes affect changes of gliatransmitters such as glutamate?Which neurons expessing NMDAR and AMPAR affected by changes in glutamate content in synaptic cleft?4.What is the effect of the altered activation status of astrocytes on the inhibitory circuitry between glycine neurons and PKCγneurons?5.Will the above changes ultimately affect the occurrence and development of neuropathic allodynia?The purpose of this study is to explore the mechanism of LTD mediated by astrocytes,in inhibitory circuits formed by Gly T2 neurons and PKCγneurons,using mouse ethology,immunofluorescence and immunoelectron microscopy,Western-blot,virus injection,calcium signal recording,DREADDs and patch clamp electrophysiology,to provide a theoretical basis for exploring the mechanism of neuropathic pain.Part one:changes of endogenous cannabinoid content around specific neurons in spinal dorsal horn after CCIObjectiveTo observe the dynamic changes of e CBs around astrocytes,PKCγneurons and Gly T2neurons in the spinal cord dorsal horn after CCI.MethodThree SPF grade male GFAP-Cre ERT2 mice,three PKCγ-i Cre mice and three Gly T2i Cre mice were selected.The GFAP-Cre ERT2 mice were injected intraperitoneally with tamoxifen to induce CRE expression.Cannabinoids probe virus was injected into the ipsilateral of CCI,ceramic core needle was fixed,and the CCI was used to establish neuropathic pain models.The fluorescence intensity of cannabinoid probe was recorded by single channel optical fiber recording system before and 4 h,8 h,1d,2 d,3 d,5 d,7 d,14 d and 21 d after CCI modeling,and then the content of e CBs was determined.ResultThe content of e CBs around GFAP cells was increased in the middle stage（2 d-5 d）of CCI modeling(middle vs.sham,One-Way ANOVA,***P<0.001).The content of e CBs around PKCγneurons was increased in all stage（4 h-14 d）of CCI modeling(vs.sham,One-Way ANOVA,*P<0.05,**P<0.01).The content of e CBs around Gly T2 neurons was increased in the early and late stage（4 h-1 d,7 d-14 d）of CCI modeling(vs.sham,One-Way ANOVA,*P<0.05,**P<0.01).ConclusionThis part of the experimental results showed that the content of endocannabinoids in the spinal cord dorsal horn increased after peripheral nerve injury,and the increase occurred in different stage after injury.The involved neurons include astrocytes,PKCγneurons and Gly T2 neurons.The unpublished data of our group also showed that AEA increased on the1st and 7th day after peripheral nerve injury,1-AG and 2-AG increased on the 7th day after injury.As the first step of our hypothesis,this part of the experiment lays a solid theoretical foundation for the follow-up research.Part two:Astrocytes in the spinal dorsal horn participate in the development of neuropathic allodyniaObjectiveTo verify the co-localization of GFAP and CB1 receptor in the spinal cord dorsal horn,and further observe the changes of pain behavior and the activation of GFAP in the spinal cord dorsal horn after CCI.GFAP-CB1-KO mice and GFAP-CB1-WT mice were used to observe the reduction of CB1 receptor on CCI induced pain behavior and the activation of astrocytes in spinal dorsal horn.GFAP were activated by chemical genetics to observe the effect on pain behavior induced by CCI.Methods1.Three SPF grade male C57BL/6 mice were used.After anesthesia,the blood was washed with PBS and then perfused with 4%paraformaldehyde.The spinal cord was fixed with 4%PFC and then dehydrated with 20%sucrose and 30%sucrose gradiently.After frozen section,3 pieces of spinal cord slices were randomly selected from each mouse,and double labeled with GFAP/CB1 immunofluorescence staining was performed to observe the co-labeling of the two in the spinal cord dorsal horn.2.Twelve SPF grade male C57BL/6 mice were selected.Six mice in CCI treatment group were given CCI model,and the other six mice in sham operation group were given sham operation.Von-Frey filament and paintbrush were used to measure the static paw withdrawal mechaical threshold and dynamic paw withdrawal mechaical score of the two groups at 6 time points before modeling,1 d,3 d,5 d,7 d and 14 d after modeling.3.Eighteen SPF grade male C57BL/6 mice were selected,with 3 mice at each time point.Immunofluorescence staining was used to observe the activation of astrocytes in the spinal cord dorsal horn before CCI modeling and on the 1st,3rd,5th,7th and 14th day after CCI modeling.4.Three GAFP-CB1-KO mice and three control GFAP-CB1-WT mice in the same nest were selected to verify whether the mice were successfully constructed by immunoelectron microscopy staining;Eight GFAP-CB1-KO mice and eight control GFAP-CB1-WT mice in the same nest were selected to measure the static paw withdrawal mechaical threshold and dynamic paw withdrawal mechaical score of the two groups at 6time points before modeling,1 d,3 d,5 d,7 d and 14 d after modeling by using Von-Frey filament and Paintbrush;18 GFAP-CB1-KO mice and 18 GFAP-CB1-WT mice in the same nest were used to observe the activation of astrocytes in the spinal cord dorsal horn before modeling,1 d,3 d,5 d,7 d and 14 d after modeling;Four groups were divided:GFAP-CB1-KO+h M3D（Gq）+CNO group,GFAP-CB1-KO+h M3D（Gq）+saline group,GFAP-CB1-KO+m Cherry+CNO group and GFAP-CB1-WT+h M3D（Gq）+CNO group.The static paw withdrawal mechaical threshold and dynamic paw withdrawal mechaical score were measured at 9 time points:0.5 h before intraperitoneal administration,0.5 h,1 h,2 h,4 h,6 h,8 h and 24 h after intraperitoneal administration on the 10th day after CCI.The pain behavior of experimental mice after astrocyte activation was observed.Results1.Cannabinoid receptor CB1 is expressed in astrocytes of the spinal cord dorsal horn.2.Stable neuropathic allodynia could be formed after CCI modeling,which was characterized by lower static paw withdrawal mechaical threshold and higher dynamic paw withdrawal mechaical score(CCI vs.Sham,Two-way ANOVA,***P<0.001,****P<0.0001);At the same time,CCI modeling also activated astrocytes in the spinal cord dorsal horn,showing an increase in the number of cells per unit area and enhanced fluorescence intensity(vs.sham,One-Way ANOVA,*P<0.05,**P<0.01,***P<0.001,****P<0.0001).3.GFAP-CB1-KO mice were successfully constructed;after the CB1 receptor on astrocytes was knocked out,the allodynia induced by CCI model was alleviated,manifested by the decrease of static paw withdrawal mechaical threshold and the increase of dynamic paw withdrawal mechanical score(KO vs.WT,Two-way ANOVA,*P<0.05,**P<0.01,***P<0.001),and the activation of astrocytes in the spinal cord dorsal horn was also reduced(KO vs.WT,Two-Way ANOVA,*P<0.05,**P<0.01)4.After CCI modeling,all experimental animals showed allodynia,and the static paw withdrawal mechaical threshold of all KO mice was higher than that of WT mice,and the dynamic paw withdrawal mechaical score of all KO mice was lower than that of WT mice(KO vs.WT,two tailed student’s unpaired t test,****P<0.0001).CCI induced allodynia was restored in GFAP-CB1-KO mice by activating astrocytes in the spinal cord dorsal horn by intraperitoneal injection of CNO.The static paw withdrawal mechaical threshold and the dynamic paw withdrawal mechaical score of the two groups were similar at some time points(GFAP-CB1-KO+h M3D（Gq）+CNO vs.GFAP-CB1-WT+h M3D（Gq）+CNO,Two-Way ANOVA,*P<0.05,****P<0.0001).ConclusionThis part of the experiment verified that CB1 receptors were expressed in GFAP cells of the spinal cord dorsal horn.Peripheral nerve injury can activate GFAP cells in the spinal cord dorsal horn and induce neuropathic allodynia in experimental animals.However,knockout of CB1 receptor on GFAP cells could reduce the CCI induced activation of GFAP in the spinal cord dorsal horn and alleviate the allodynia induced by CCI.Chemogenetic activation of astrocytes in GFAP-CB1-KO mice restored CCI induced allodynia.It is fully proved that astrocytes play an important role in the development of neuropathic allodynia.This study provides a theoretical basis for the further study of the function of spinal inhibitory circuits.Part 3:Astrocytes mediated LTD of inhibitory circuits between Gly T2neurons and PKCγneurons in the spinal cord dorsal horn participate in the development of neuropathic allodyniaObjectiveTo investigate the role of astrocytes mediated LTD of inhibitory circuit between PKCγneurons and Gly T2 neurons in the formation of neuropathic allodynia.Methods1.Eighteen SPF grade male C57BL/6 mice were selected and perfused with 0.9%normal saline on the 1st,3rd,5th,7th and 14th day after CCI modeling,and then the ipsilateral lumbar enlargement was taken for protein quantitative test to observe the content of Glu R2 in surface membrane protein and total membrane protein.Thirty two mice were given intrathecal administration before and after CCI to observe the effect of interference with Glu R2 endocytosis on pain behavior.2.Six PKCγ-td Tomato mice and six Gly T2-td Tomato mice were used to observe the endocytosis of Glu R2 before and after CCI by immunoelectron microscopy,and to determine which neurons were involved in the endocytosis.3.Six PKCγ-td Tomato mice and six Gly T2-td Tomato mice were used to observe the effects of perfusion drugs on the electrical activity of corresponding neurons after Glu R2endocytosis by whole cell patch clamp recording method.4.Six Gly T2-td Tomato mice were used to observe the effect of CCI on the excitability of Gly T2 neurons.5.24 PKCγ-i Cre mice and 24 Gly T2-i Cre mice were used to inhibit the activity of PKCγneurons or activate the activity of Gly T2 neurons by chemical genetic method,and the effect on pain behavior of mice was observed.Results1.The expression of membrane Glu R2 was significantly decreased from 3 to 14 days after CCI(vs.sham,One-Way ANOVA,*P<0.05,**P<0.01).However,the total expression of Glu R2 did not change（vs.Sham,P>0.05,）,indicating that the endocytosis of Glu R2 on the membrane of spinal cord neurons occurred after CCI modeling;Intrathecal administration of Tat-Glu R2 to interfered the endocytosis of Glu R2,which could delay CCI induced neuropathic allodynia,manifested as slow decline of static paw withdrawal mechaical threshold and slow rise of dynamic paw withdrawal mechanical score,and alleviated the neuropathic allodynia(CCI+Tat-Glu R2 vs.CCI+DDH2O,Two-way ANOVA,*P<0.05,**P<0.01,***P<0.001,****P<0.0001).2.After CCI modeling,Glu R2 on the membrane of Gly T2 neuron was internalized(Gly T2-sham vs.Gly T2-CCI,chi-square test,****P<0.0001).3.The inhibition of Glu R2 endocytosis by perfusion of Tat-Glu R2 partially reversed the effect of LFS on LTD of e IPSPs recorded in PKCγneurons,but not by perfusion of Tat-Glu R2S(LFS+Tat-Glu R2 vs.LFS,Two-way ANOVA,*P<0.05,**P<0.01,****P<0.0001).4.After CCI modeling,the proportion of burst action potential of Gly T2 neurons and the amplitude of action potential decreased significantly(Pre-CCI vs.Post-CCI,65.2%vs33.3%,two tailed student’s unpaired t test,**P<0.01).In addition,the discharge threshold and basal intensity of Gly T2 neurons were also higher than those of normal mice(Pre-CCI vs.Post-CCI,two tailed Student’s unpaired t test,**P<0.01,***P<0.001).5.Injection of h M4Di into spinal cord dorsal horn and intraperitoneal injection of CNO to inhibite PKCγneurons can increase the static paw withdrawal mechaical threshold induced by CCI(vs.h M4Di+saline,Two-way ANOVA,****P<0.0001,vs.EYFP+CNO,####P<0.0001),reduce the paw withdrawal mechanical score(vs.h M4Di+saline,Two-way ANOVA,*P<0.05,**P<0.01,***P<0.001,vs.EYFP+CNO,###P<0.001,####P<0.0001),and increase the threshold of Hargreaves(vs.h M4Di+saline,Two-way ANOVA,****P<0.0001,vs.EYFP+CNO,##P<0.01,###P<0.001,####P<0.0001);The activation of Gly T2 neurons in the spinal cord dorsal horn by injection of hm3Dq virus into the spinal cord dorsal horn and intraperitoneal injection of CNO increased the static paw withdrawal mechaical threshold induced by CCI(vs.h M3Dq+saline,Two-way ANOVA,***P<0.001,vs.EYFP+CNO,###P<0.001),reduce paw withdrawal mechanical score(vs.h M3Dq+saline,Two-way ANOVA,**P<0.01,***P<0.001,****P<0.0001,vs.EYFP+CNO,##P<0.01,###P<0.001),and increase the threshold of Hargreaves(vs.h M3Dq+saline,Two-way ANOVA,**P<0.01,***P<0.001,****P<0.0001,vs.EYFP+CNO#P<0.05,##P<0.01,###P<0.001).ConclusionThe endocytosis of Glu R2 occurred in the spinal cord dorsal horn after CCI,and this endocytosis occurred in Gly T2 neurons.Intrathecal administration of Tat-Glu R2 interferes with the endocytosis of neuron membrane surface protein Glu R2,which can delay the neuropathic allodynia induced by nerve injury and alleviate the established allodynia.Inhibition of Glu R2 endocytosis by perfusion Tat-Glu R2 reversed the LTD induced by LFS stimulation in PKCγneurons.In addition,the activity of Gly T2 neurons decreased after CCI.Inhibition of PKCγactivity or activation of Gly T2 activity can alleviate the pain behavior of the experimental mice.Summary1.The content of endocannabinoids increased around astrocytes,PKCγneurons and Gly T2 neurons in the spinal cord dorsal horn after nerve injury.2.After CCI,the increased endocannabinoids activated astrocytes by acting on CB1receptor.Knockout of CB1 receptor on astrocytes could alleviate CCI induced neuropathic allodynia.Reactivation of astrocytes in GFAP-CB1-KO mice may aggravate CCI induced neuropathic allodynia.3.Glu R2 endocytosis occurred in Gly T2 neurons of spinal dorsal horn after CCI.Drug interference with Glu R2 endocytosis could alleviate neuropathic allodynia induced by CCI and reversed LTD induced by LFS stimulation on PKCγneurons.In addition,the disinhibitory activity of PKCγneurons increased,while the excitability of Gly T2 neurons decreased after nerve injury.Inhibition of PKCγneurons or activation of Gly T2 neurons can alleviate CCI induced neuropathic hyperalgesia.Overall conclusionAfter nerve injury,the content of endocannabinoids around astrocytes、PKCγand Gly T2 neurons increased,and the activation of astrocytes by CB1 receptor causes the occurrence and maintenance of neuropathic allodynia.Activated astrocytes release glutamate and act on Gly T2 neurons in spinal cord dorsal horn to induce Glu R2endocytosis,which in turn affects the activity of downstream PKCγneurons.In addition,nerve injury can also affect the innatequality of PKCγneurons and Gly T2 neurons.And the regulation of these two types of neurons can affect the neuropathic allodynia induced by nerve injury.In this study,we found that astrocytes and their CB1 receptors were vital for neuropathic allodynia.In addition,PKCγneurons and Gly T2 neurons also participate in the development of allodynia,which provides the possibility to clarify the mechanism of neuropathic allodynia and explore effective therapeutic targets. |
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