| Background:Irritable bowel syndrome(IBS)is a global epidemic functional gastrointestinal disease,and its main symptoms include constipation,diarrhea,and abdominal pain.About 10-25%of the world’s population is affected by IBS.Chronic visceral pain is a type of IBS,owing to the lack of clinical samples of patients with IBS and lack of effective treatments in clinical,finding a new therapeutic targets is critical.Therefore,a model of Neonatal Maternal Deprivation(NMD)was prepared to simulate chronic visceral pain in patients with IBS.Using optogenetics,fiber Photometry,chemgenetics,gene transcription and protein expression detection,the role of NR2A in glutamatergic neurons of the VTA in the development of chronic visceral pain was investigated.Methods:1.Establishment of neonatal maternal deprivation(NMD)model:The experimental animals used in this study were C57BL/6J mice.The pups were separated from their mothers 3 hours per day from the second day after birth for continunous 14 days,while CON mice did not receive any treatment.Experiments were performed when the mice were 6 weeks old.2.Colorectal distention(CRD):The expandable balloon was made from the small finger portion of a small latex glove and a thin tube,and then inserted it from the mouse’s anus to the colorectal region.The pain threshold of mice was detected by giving different degrees of dilation pressure through the sphygmomanometer and combining with the area under the curve measured by the electromyography.3.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR):The expression of NR2A in the VTA of CON and NMD mice was measured at the mRNA level.4.Western Blotting:The expression of NR2A in the VTA of CON and NMD mice was detected at the protein level.5.Immunofluorescence:The expression of c-Fos and neuron types of c-Fos labeled nerouns in VTA were detected in the CON group,the CON+CRD group,the NMD group,and the NMD+CRD group.At the same time,the c-Fos expression of VTA was detected after injecting NR2A antagonist NVP-AAM077 and the same dose of normal saline when giving stimulation with CRD.6.Optogenetics:The blue light photoactive virus of AAV-VGLUT2-hCHR2-EGFP was injected into VTA of CON mice,and the yellow light photoinhibtion virus of AAV-VGLUT2-eNpHR-EGFP was injected into the VTA of NMD mice.The visceral pain changes of mice were detected by before and after different wavelengths of light to stimulate VTA glutamatargic neurons specifically activate or inhibit.7.Fiber Photometry:The AAV-VGLUT2-GCamp6s virus was injected into VTA of CON and NMD mice.The change of glutamatargic neurons calcium signal intensity was monitored and recorded in real time.8.Chemgenetics:The chemical genetically activated virus AAV-VGLUT2-hM3DqmCherry was injected into VTA to allow specific activation of glutamatergic neurons,and the changes of visceral pain were compared before and after activation.Results:1.CRD-induced the expression of c-Fos in the VTA neurons of the NMD group was significantly increased compared to the CON group,and most of them were colabeled with glutamatergic neurons.2.The optical fiber recording experiments revealed that the calcium signal of VTA glutamategic neurons in response to CRD was significantly enhanced compared to the CON mice.3.Compared with non light stimulation,optogenetic inhibition of VTA glutamatergic neurons significantly reduced the AUC in NMD mice,indicating a relief of visceral hyperalgesia in NMD mice.4.Optogenetic activation of VTA glutamatergic neurons significantly increased the AUC in CON mice,indicating development of visceral hyperalgesia in CON mice.5.Compared to CON mice,the expression of NR2A receptor subunits in VTA of NMD mice were significantly increased at both mRNA and protein level.6.Comapred with the same dose of normal saline group,the visceral pain threshold of NMD mice was significantly increased after injecting NR2A antagonist NVPAAM077 into the VTA,which relieved the visceral pain of NMD mice.In a certain concentration range,the duration of the effect was enhanced with the increase of drug dosage concentration.At the same time,the expression of c-Fos in VTA was significantly decreased in NMD mice injected with NR2A antagonist NVPAAM077.7.Chemical activation of VTA glutamatergicgic neurons in CON mice significantly increased visceral pain response,indicating visceral hyperalgesia,which was reversed when NR2A antagonist NVP-AAM077 was injected into VTA.8.The relieving effect of NR2A antagonist NVP-AAM077 on visceral heperalgesia of NMD mice was reversed by chemical activation of VTA glutamatergicgic neurons.Conclusions:This study employing the state-of-art techniques in neuroscience research demonstrated that the up-regulated expression of NR2A receptor subunit in VTA glutamatergic neurons of NMD mice mediated enhanced excitability of glutamatergic neurons,thus inducing chronic visceral hyperalgesia in NMD mice.In conclusion,NR2A in VTA glutamatergic nerouns may be the key molecule involved in chronic visceral hyperalgesia of NMD mice,providing a new target for the development of drugs therapies for chronic visceral pain. |
