Dopamine Receptor D2 Mediates Reward Circuit From VTA To CeA-produced Pain Relief

BackgroundPain is a physically and emotionally debilitating condition which is a multidimensional conscious experience including sensory and negative affective components.The negative affect including anxiety and depression is well known to accompany the pain perception during disease progression,exerting huge physical and mental burden on the patients.Up to now,pain is complex and intractable.Pain sensation consists of two parts:pain sensation and pain emotion.Among them,pain sensation is mainly judged by the nature,intensity and location of the stimulus,and pain sensation is mainly the unpleasant feeling accompanying pain.However,mechanisms underlying the negative affects of pain have not been fully identified.It may provide a new target in the future for clinical.Pain relief has the property of reward.The classic reward circuit begins in the ventral tegmental area of the midbrain,which is rich in dopamine neurons,and ends in the nucleus accumbens(NAc)in the forebrain.DA neurons originated from VTA have a wide range of projections.These neurons play a pivotal role in pain regulation through projecting to the hippocampus,olfactory lobe,prefrontal cortex and amygdala.The amygdala,as the subcortical center of the limbic system,plays a key role in pain modulation.In particular,the central amygdala(CeA),also known as the"injury-sensing amygdala",has a strong sensory and emotional dimension.In the meanwhile,neurotransmitters,neuropeptides,hormones and corresponding receptors are involved in pain regulation of the amygdala together.As an important neurotransmitter in the midbrain reward system,DA plays a role through the corresponding DA receptor.Several studies have shown that dopamine works through five different G protein-coupled receptors,including the D1 subfamily(D1 and D5)and the D2 subfamily(D2,D3 and D4).The former is coupled with Gs and promotes the formation of cAMP,while the latter is coupled with Gi and inhibits the formation of cAMP,shown that dopamine receptors play different roles in regulating nociception in different nuclei in the brain.Therefore,this subject established a model of plantar incision pain relief.Using optogenetics technology combined with behavioral testing of conscious animals,it is revealed that the specific activation of the VTA-CeA reward pathway is involved in pain relief.What's more,the dopamine D2 receptor of the amygdala(D2R)mediates the mechanism of the VTA-CeA pathway to relieve pain sensation pain emotion.ObjectiveThis study aimed to investigate whether activation of VTA-CeA reward pathway can relieve pain sensation and pain emotion,and whether the D2R mediates VTA-CeA pathway in alleviating pain sensation and pain emotion.MethodsIn this study,we established a plantar incision-induced pain model in mice,and detected the behavioral changes of the mice postoperatively.Popliteal injection of Lidocaine was found to alleviate pain in mice(producing conditional position preference).Immunofluorescence was performed to detect the expression of the fos in the VTA,which to verify the activation of the VTA in the pain relief.The fiber projection was performed by tracer technique and immunofluorescence double standard.Then,we detected the changes of the pain sensation and emotional behavior when the VTA-CeA pathway was activated by optogenetics in mice.Western blot was used to determine the expression of dopamine D1 and D2 receptors in the CeA when the pain relief.Then we detected the double labeling of D1R,D2R,GABA and Glutaminase.Finally,we observed the changes of pain sensation and pain emotion after administering D2R agonist.1.Establishment of the planter incision-induced pain and its relief model and the pain sensation was detected by Von Frey and thermal withdrawal latency,the pain emotion was detected by the open field and elevated plus maze experiment.Mice were anesthetized with isoflurane.The left hind foot of the animal was sterilized with iodine voles,and a longitudinal incision of 0.5 cm was made through the skin,fascia and plantar muscle.Pressure was applied to stop hemorrhage and the skin was sutured.Sham operation group only received anesthesia and disinfection.Mechanical pain and thermal pain were detected prior to operation,6 hours,1st day,3rd days,and 7th days post the operation.Anxiety-like emotions were detected on 3rd day post operation.Peripheral nerve block(PNB)was performed by injecting 100ul normal Saline or Lidocaine(2%)into the popliteal fossa under isoflurane mild anesthesia,and conditioned positional preference was detected in the mice.2.Immunofluorescence was used to detect the activation of neurons in the VTA region during pain relief.Tracer technique combined with immunofluorescence was used to detect the projection between dopaminergic neurons in the VTA region and CeA.In normal male BL/6J mice,100ul of normal Saline or Lidocaine(2%)was injected into the popliteal fossa under isoflurane mild anesthesia to achieve peripheral nerve block(PNB).Immunohistochemistry was performed to measure the expression of Fos in the VTA region in sham+Saline,sham+Lidocaine,incision+Saline,and incision+Lidocaine groups.Normal male BL/6 j mice under isoflurane anesthesia,were fixed on the brain stereotactic apparatus,and craniotomy was performed.1ul Hamilton with glass electrode micro syringe was used to inject retrograde tracer fluorescence gold FG(2%,diluted Saline)in CeA area(AP 1.22 mm,ML.2.3 mm,DV.4.8 mm),after which FG positive neurons in VTA region was observed.Alternatively,the AAV8-EF1a-DIO-ChR2-mCherry virus was injected in the VTA area to observe mCherry positive nerve fibers in the CeA area.To prevent leakage,the needle can be retained in situ for 30 min after the drug injection before being slowly pulled out.The projection in perfusion section was observed directly under fluorescence microscope 7?10 days post modeling.The co-expression of FG and TH was detected by immunofluorescence technique.3.The VTA-CeA pathway was activated by optogenetics to detect the changes of pain sensation and pain emotion.Firstly,AAV8-EF1a-DIO-ChR2-mCherry or AAV8-EF1a-DIO-mCherry were injected into the VTA area of DAT-Cre mice.After 4 weeks,optical fiber was embedded in CeA.The threshold baseline of the mice was detected 7 days later to exclude the effects of optical fiber placement.Planter incision was performed in qualified pain model.Behavioral detection was detected in the state of blue light activation.After the experiment,perfusion was used to detect whether the light-sensitive protein was expressed on dopamine neurons by immunofluorescence technique.4.Western blot was performed to dectect the expression alteration of dopamine receptors during pain relief.Intraperitoneal injection of D2R agonist combined withoptogenetics was used to further demonstrate changes in the pain sensation and pain emotion.Normal male BL/6J mice were divided into 4 groups,including sham+Saline,sham+Lidocaine,incision+Saline,and incision+Lidocaine groups.Western blot were used to detect the expression the change of DIR and D2R in CeA region.double Immunofluorescent labeling methods was used to identify the co-expression of D1R,D2R,GABA,and Glutaminase.Finally,D2R agonist was administered to the abdominal cavity to detect the changes of pain sensation and pain emotion after light activation.Results1.Mechanical pain sensitivity,thermal pain sensitivity and anxiety-like emotions were induced in the pain model of incision.In the sham group,there was no significant change in every time point after operation compared to the base value(P>0.05).However,the pain threshold of the surgical side of the model group(incision)was significantly decreased 6 hours after the incision and lasted for 3 days(***P<0.001).On 3rd day post operation,results of the open field experiment and the elevated plus maze experiment illustrated the appearance of anxiety-like behaviors.2.VTA neuron activation increases while pain was relieved,and VTA dopaminergic neurons projected to CeA.Normal male BL/6J mice were given 100ul Saline or Lidocaine(2%)in the popliteal fossa under isoflurane mild anesthesia to achieve peripheral nerve block(PNB).The results showed that the lasting time of popliteal incision+Lidocaine in plantar incision pain-induced mice was significantly higher than those of incision+Saline,indicating that CPP was induced and pain was relieved(**P<0.01,).Then the expression of fos in VTA of sham+Saline,sham+Lidocaine,incision+Saline,incision+Lidocaine was detected by immunofluorescent double-labeling method.Results showed that the expression of c-fos in VTA region of incision+Saline group was significantly increased compared to incision+Lidocaine group(*P<0.05).However,there was no significant change in the expression of c-fos in VTA region in two groups(P>0.05).Fluoro-gold(FG),a retrograde tracer,was injected into CeA in mice.Section can be directly observed under the microscope.The results showed that FG positive neurons were mainly on the same side of VTA.Anterograde virus results showed that there were a large number of mCherry positive nerve fibers in the CeA region.Immunofluorescence double-labeling technique showed that FG and TH(dopamine neuron marker)had a large number of co-labeling,and results showed that VTA and CeA had direct projection and were dopaminergic neurons combined with ante-retrograde tracer.3.Activation of the VTA-CeA pathway could relieve the mechanical pain sensitivity and anxiety-like behavior of pain mice,and produced conditional positional preference.The virus injection was completed 4 weeks later and the fiber was placed in the CeA zone.Behavioral tests were carried out after the animals survived for 1 week.The results showed that in the mice injected with AAV-ChR2 virus,the mechanical pain threshold of the posterior foot on the operative side significantly increased(**P<0.01)when compared to the light stimulation on the operative side(**P<0.01).In the open field and elevated plus maze experiments,in the AAV-ChR2 virus group,after the injection of light stimulation,the percentage of the total time(center time%)of the mice's stay time in the central region increased significantly(**P<0.01).In the conditional position preference experiment,it was shown that in the AAV-ChR2 group,the residence time of the luminal matched with light stimulus was significantly higher than that of the luminal unlit stimulus(**P<0.01).In summary,the results showed that activation of the VTA-CeA pathway could relieve the mechanical pain sensitivity and anxiety-like behavior of the incision pain mice,and generate conditional position preference.4.Intraperitoneal injection of D2R agonist reversed the light-activated VTA-CeA pathway in the relief of pain sensation and pain emotion in incision pain mice.Western blot were used to detect the protein expression of D1R and D2R in CeA region in normal male BL/6J mice,sham+Saline,sham+Lidocaine,incision+Saline,and incision+Lidocaine.The results showed that the expression of D2R in CeA was significantly down-regulated(**P<0.01),and the expression of D1R was not significantly changed(P>0.05).Results of immunofluorescence showed that D1R was mainly co-expression with Glutaminase,and D2R was mainly co-labeled with GABA.After 10 minutes of D2R agonist administration,compared to D2R+quinpirole and D2R+DMSO,the mechanical pain threshold was significantly reduced(**P<0.01).Compared to D2R+quinpirole and D2R+DMSO,the percentage of the central region residence time in the total time decreased significantly(**P<0.01).Compared to the quinpirole chamber residence time,DMSO matching chamber residence time significantly increased in light stimulation.These results suggested that D2R agonists reverse the pain relief induced by photoactivation of the VTA-CeA pathway.ConclusionReward pathway VTA-CeA is involved in relieving pain sensation and pain emotion when it is activated;D2R mediates the regulation mechanism of the VTA-CeA pathway to alleviate pain sensation and pain emotion.