A Study Of Dopamine D2 Receptors In The Anterior Cingulate Cortex Participating In The Regulation Of Pain Aversion In Rats

Objective: As one of the main clinical manifestations and complications of many diseases,pain can be divided into acute pain and chronic pain.Among them,chronic pain is also a disease,which seriously affects the quality of life of patients and the benefits of social production.The latest definition of pain includes fourdimensions: sensation,emotion,cognition and social component.The negative aversional experience,such as disgust and unhappiness,accompanied by pain,brings more pain to patients than the pain itself.The sensory discrimination and aversional experience components of pain are respectively mediated by the lateral sensory pathway and the medial aversional pathway,the medial pain nerve pathway ends in the anteriorcingulate cortex(ACC).ACC,especially the rostral ACC(rACC),has been reported many times on the regulation of pain.Ourprevious work has shown that activating rACC opioid receptors can inhibit the activity of NMDA and relieve pain in rats.And otherstudies have shown that central dopamine is anotherimportant pain modulatorbesides opioids.ACC receives dopaminergic projections from the ventral tegmental areas(VTA)and has dopamine D1 and D2-like receptors,but in the rodent neuropathic pain model,the D2 receptors of ACC has a more concentrated and effective regulation of pain than the D1 receptors.In otherbrain areas outside of ACC,activation of dopamine D2 receptors can produce significant analgesic effects,and inhibition of D2 receptors can cause nociceptive effects.However,whetherthe dopamine D2 receptors in ACC can regulate pain aversion in rats is still unclear. In this study,rat models of inflammatory pain were induced by complete Freund’s adjuvant(CFA),used conditioned place avoidance(CPA)and open field test(OFT)behavioral paradigms,combined with multi-channel electrophysiological technology,immunofluorescence technology,and behavioral testing methods of mechanical pain and thermal pain,to explore whetherdopamine D2 receptors of ACC are participate in the regulation of pain aversional in rats Methods:1.Establishment of inflammatory pain model and conditional position avoidance(CPA)model a)The model of inflammatory pain: Injectied 0.08 ml of CFA into the left hind paw of the rat,and measured the paw withdrawal mechanical threshold(PWMT)and paw withdraw thermal latency(PWTL)of the rats with a dynamic plantartactile device and a thermal radiometerrespectively,to determine the rat model of inflammatory pain was successfully established. b)The model of CPA: It is a classic paradigm forevaluating negative aversional of rats.The self-made modified conditional position avoidance device was used to monitorthe staying time of rats on the “non-pain” side and “pain” side to determine the successful,reflecting the CPA behaviorinduced by CFA,namely C-CPA.2.Groups of animals a)During the pain modeling and CPA modeling: the experimental rats were divided into three groups: Naive,saline,and CFA. b)During the formal experiment: Sham group(without treatment of paws and rACC);CFA-drugs gruops: CFA was injected into the left hind paws,and the normal saline(NS)/Quinpirple/Eticlopride were injected into rACC;NS-drugs groups: NS was injected into the paws,and NS / Quinpirple / Eticlopride were injected into rACC,n=8～10.3.Injection of dopamine D2 receptoragonists orantagonists into rACC areas a)Embedding the drug delivery tubes: The self-made drug delivery tubes were surgically embedded in the rACC of in rats brain. b)Injecting drugs into rACC: Injected dopamine D2 receptoragonist orantagonist into the rACC of rats with CFA-induced pain orNS-treated control rats through an electronic pusherpump.4.To detect whetherdopamine D2 receptors can regulate pain aversion in rats Detected the changes in CPA of experimental rats afterreceiving corresponding treatments(Sham,CFA-NS,CFA-Quinpirple,CFA-Eticlopride,NS-NS,NS-Quinpirple,NS-Eticlopride)to determine the effects of rACC DRD2 on pain aversion in rats.5.The open field test comprehensively evaluated the effects of negative aversion and movement ability that activatied orinhibited DRD2 on rACC areas. Detected the time spent in the open field centerof seven groups(Sham,CFA-NS,CFA-Quinpirple,CFA-Eticlopride,NS-NS,NS-Quinpirple,NS-Eticlopride)of experimental rats,reflecting the changes in pain-related anxiety-like aversions.The total movement distance during the test time was counted to evaluate the movement ability changes of rats afterbeing treated with corresponding drugs.6.Filtered out the pyramidal neurons and interneurons in the ACC brain area based on the discharge characteristics of different neuron types observed in the process of multi-channel electrophysiological recording According to the typical firing characteristics of pyramidal neurons and interneurons,the pyramidal neurons in ACC brain areas of each group were selected,and the firing activities of ACC neurons in all groups were counted on this basis.7.Using multi-channel electrophysiological technology,synchronously monitorthe firings of neurons in the ACC brain areas during the behavioral response of CPA in rats,and explore the regulation of DRD2 on CPA response. a)Fabrication of microwire electrode arrays and embedded them. b)Using multi-channel electrophysiology to detect the discharge frequency of pyramidal neurons in ACC brain areas on the “non-pain” side and “pain” side in the Naive,NS and CFA groups of rats,to provide objective and intuitive evidence forC-CPA behaviors in painful rats. c)Recording the electrical signals pyramidal neurons in ACC brain areas Sham,CFA-NS,CFA-Quinpirplre,CFA-Eticlopride,NS-NS,NS-Quinpirplre and NS-Eticlopride groups on the “non-pain” side and “pain” side,objectively reflected the regulating effect of D2 receptoron pain in rats8.Immunofluorescence Immunofluorescence double-labeling experiments of dopamine D2 receptors and Glu N1 subtype of NMDA receptors on layerII/III neurons of rACC.Results:1.Successfully induction pain and C-CPA model a)Compared with the NS group and the Naive group,the PWTL and PWMT of rats in the CFA group were significantly reduced(P<0.001). b)Compared with the NS and Naive groups,the rats in the CFA group stayed on the“non-pain side” significantly longerthan the “pain” side(P<0.01).2.Injection of dopamine D2 receptoragonist Quinpirple into rACC could effectively alleviate C-CPA behaviorin rats In the CFA-induced pain model rats,equal volume of NS orQuinpirple were injected into rACC.Compared with CFA-NS groups,the rats in the CFA-Quinpirple group stayed on the “pain” side significantly longer(P<0.05);3.Injection of dopamine D2 receptorantagonist Eticlopride into rACC could furtheraggravate C-CPA behaviorin rats In the CFA-induced pain model rats,equal volume of NS orEticlopride were injected into rACC.Compared with CFA-NS groups,the rats in the CFA-Eticlopride group stayed on the “pain” side significantly shorter(P<0.05)4.Injection of dopamine D2 receptoragonist orantagonist into rACC would not change the staying time of the rats injected with NS on the “pain” side Injected 0.08 ml NS into the left hind paws of the rats,and injected 10 (?)g Quinpirple orEticlopride orthe corresponding volume of NS into rACC.There were no significant difference in the staying time of the rats in the NS-Quinpirple and NS-Eticlopride groups on the “pain” side.5.Open field test results Activation orinhibition of DRD2 of rACC areas would change the time spent in the centerof the open field in rats with inflammatory pain,but would not change the total movement distance.But activating orinhibiting DRD2 of rACC areas would not change the time spent in the centerand the total movement distance of rats injected with NS.6 Multi-channel electrophysiology mainly recorded the firings of pyramidal neurons In the process of multi-channel recording,it was obvious that most of the neurons recorded by the channels show typical firings characteristics of pyramidal neurons7.Multi-channel detection of the excitability of pyramidal neurons in the ACC brain area of CFA-induced pain model rats in the “pain ” side Using multi-channel electrophysiological technology,rats were used to detect the discharge activity of pyramidal neurons in ACC brain areas on the “pain” side and“non-pain” side for10 minutes on each side.Aftercomparison,the discharge frequency of rats in the Naive and NS-NS groups was not significant on both sides.The difference was that the firings frequency of rats in the CFA-NS group on the “pain” side was significantly increased,indicating that the excitability of rACC neurons of rats with pain induced by CFA was increased,which furtherverified the C-CPA behaviorand provided objective evidence forthe generation of pain aversion.8.Activation of rACC D2 receptors can restore the discharge frequency of pyramidal neurons in the ACC brain area of CFA-induced pain model rats in the “pain ” side Compared with the CFA-NS groups,the discharge frequency of pyramidal neurons in ACC brain areas of CFA-Quinpirple group on the “pain” side was significantly reduced(P<0.05),indicating that the pain aversion of the rats were effectively relieved afterinjection of Quinpirple into rACC.9.Inhibition of rACC D2 receptors can furtherincrease the discharge frequency of pyramidal neurons in the ACC brain area of CFA-induced pain model rats in the “pain ”side Compared with the CFA-NS group,the discharge frequency of pyramidal neurons in ACC brain areas of CFA-Eticlopride group were significantly higheron the “pain” side(P<0.05),indicating that the aversion of rats with pain was furtherexacerbated afterinjection of Eticlopride into rACC.10.Activating orinhibiting DRD2 in rACC does not change the excitability of pyramidal neurons in the “pain ” side of rats injected with NS on the paws Compared with NS-NS group,the discharge frequency of pyramidal neurons in ACC brain areas of NS-quinpirple and NS-epiclopride groups had no significant change in the “pain” side,indicating that injection of Quinpirple orEticlopride into rACC did not affect neuronal excitability.11.Injection of rACC with agonists orantagonists of D2 receptors would not change the PWTL and PWMT of rats Compared with the control group,rACC injection of Quinpirple and Eticlopride did not change the PWTL and PWMT,and did not reverse orchange the pain sensation.12.Co-expression of dopamine D2 receptors and NMDAR1 receptors in the neurons of layerII/III of rACC Because there are a large numberof pyramidal neurons in the Ⅱ/Ⅲ layerof ACC,this part was selected forimmunofluorescence double labeling.The results showed that there was a large amount of co-localization of dopamine D2 receptors and Glu N1 receptors,a subtype of NMDA receptorin the target field of view.Conclusion:Activation of dopamine D2 receptors in rACC brain areas could reverse the C-CPA response and reduce the discharge activities of pyramidal neurons on the “pain” side of rats;Inhibition of dopamine D2 receptors in rACC brain could furtherincrease the C-CPA behaviorand increase the discharge activities of pyramidal neurons in the “pain”side of rats;Dopamine D2 receptors in the brain areas of rACC might participate in the regulation of pain aversion during the inflammatory pain induced by CFA.