| Objective:As a major clinical problem in today’s society,pain has received increasing attention.According to the latest definition of pain by the International Society for the Study of Pain（IASP）,pain can be divided into four major aspects:the perception of pain,the emotional response to pain,the cognitive process of pain,and the social dimension.Anterior cingulate cortex（ACC）is an important part of the brain’s limbic system that is involved in emotion,memory and behavior.Substantial evidence from clinical and experimental studies indicates that the anterior cingulate cortex（ACC）plays a role in the management of harmful stimuli and pain-related depression and anxiety.Multiple studies have shown that ACC is involved in coding the emotional aspects of pain.NMDA receptors play important roles in synaptic transmission,long-term enhancement,learning and memory,epilepsy,pain,and are also abundantly expressed in the ACC area.NMDA receptors are also involved in painful emotional responses.Opioid analgesics are widely used as traditional analgesics,but they also have many side effects.There are mainlyμ-,δ-,andκ-opioid receptors in the ACC region.In our study,we chose DAMGO,which is a synthetic opioid peptide that exhibits high selectivity forμ-relative toδ-andκ-opioid receptors.Our previous research has confirmed that after CFA injection,NMDA receptors are activated and pain is aggravated.In addition,in vivo activation of opioid receptors can alleviate pain in rats,but the relationship between opiate receptors and NMDA receptors is still unclear.This study will explore this issue in depth.The animal model used in this experiment was a model of inflammatory pain induced by complete Freund’s adjuvant（CFA）injection in the soles of rats,and then an isolated brain slice patch clamp test was performed to detect NMDA current.To investigate whether the activation ofμ-opioid receptors in rACC can inhibit the effect of NMDA current in this experiment.Methods:1.CFA-induced pain animal model2-3 weeks of male SD rats were randomly divided into two groups:the left hind paw was injected subcutaneously with Freund’s adjuvant（CFA）0.08ml group and the left hind paw was injected subcutaneously with the same amount of normal saline（NS）group.2.Measure the thermal contraction latency（PWL）of the two groups of rats3.Whole-cell patch-clamp experiments on isolated brain slicesAfter the rats were anesthetized,the brain tissue was taken out,cut into 300μm tissue slices using a vertical vibration microtome,and incubated for one hour at room temperature in an artificial incubation solution for recording of whole-cell patch clamp experiments.（1）A brain slice patch-clamp system with an infrared differential interference phase contrast microscope was used to select and identify pyramidal neurons in a whole-cell mode;（2）Set the clamping voltage to-60 mV,and record excitatory post-synaptic current of neurons in rACC area;（3）Set the clamping voltage to+40 mV,record the NMDA current of neurons in the rACC area,and give the NMDA receptor antagonist AP-V for verification;（4）Set the clamping voltage to+40 mV,record the NMDA current of neurons in the rACC region,and verify the AP-V of the NMDA receptor antagonist,ACSF elutes for 10 minutes,the effects of different concentrations ofμopioid receptor agonist DAMGO（10-1212 mol/L,10-99 mol/L,10-66 mol/L）on the NMDA current were recorded in the perfusate.Results:1.CFA injection in rat hind paw can produce inflammatory pain responseOn the third day,the PWL value of the rats in the CFA injection group was significantly shortened compared with its basic value（P<0.05,n=5）.On the third day,the PWL value of the rats in the NS group was not significantly different from its basic value（P>0.05,n=5）.This shows that CFA injection on the left hind paw can cause persistent inflammatory pain in rats.2.Morphological results of neurons in rACC brain areaThe basic morphological characteristics of neurons in rACC brain region can be clearly observed by infrared differential interference phase contrast microscope and fluorescence microscope,including the cell body,axon.3.rACC brain area electrophysiological results（1）The neurons selected for recording belong to pyramidal neuronsIn the whole-cell voltage-clamp recording mode,a significant inward sodium current was recorded after depolarization stimulation was applied,indicating that the recorded cells were neurons.（2）The injection of CFA on the plantar leads to an increase in the frequency of excitatory postsynaptic current（EPSC）in neurons of rACC brain region.Compared with the plantar injection NS group,the frequency of excitatory postsynaptic current（EPSC）in neurons of rACC brain region of plantar injection CFA group increased significantly（P<0.05,n=5）.This indicates that plantar injection of CFA can increase neuronal excitability in rACC brain regions.（3）CFA injection on the soles of the feet leads to an increase in the amplitude of NMDA currents in rACC brain neurons.Compared with the plantar injection NS group and the plantar injection CFA group,the NMDA current amplitude significantly increased（P<0.05,n=5）.These results indicate that NMDA receptors can be activated after injection of CFA on the plantar floor of rats,which increases the channel opening.（4）The plantar injection of CFA did not significantly change the amplitude of AMPA currents in rACC neurons.Compared with the plantar injection NS group and the plantar injection CFA group,the amplitude of AMPA current did not change significantly（P>0.05,n=5）.It indicated that the injection of CFA in rats did not activate AMPA receptors,and AMPA receptors were not involved in the process of chronic inflammatory pain.（5）Perfusion ofμopioid receptor agonist DAMGO in rat brain slices can inhibit the increase of NMDA current caused by CFATo further clarify the interaction between theμopioid receptor and NMDA receptor in the rACC region,we chose to add theμopioid receptor DAMGO to the perfusate.Compared with the CFA injection group,different concentrations ofμopioid receptor agonists have different inhibitory effects on NMDA current.There was no significant difference in current amplitude between the perfusion 10-12μmol/L DAMGO group and CFA group（P>0.05,n=5）.The current amplitude of the perfusion group of10-9μmol/L DAMGO was significantly reduced（P<0.05,n=5）,and the current of the perfusion group of 10-6μmol/L DAMGO was almost completely suppressed（P<0.05,n=5）,with the strongest inhibition effect.This shows that the opioid agonist DAMGO can inhibit NMDA current,and there is a dose-dependence between different concentrations.（6）DAMGO,a mu opioid receptor agonist perfused into rat brain slices,has no effect on AMPA currentSimilarly,we also observed the effect of theμopioid receptor agonist DAMGO on AMPA current.The results showed that compared with the CFA group,different concentrations ofμopioid receptor agonists had no significant inhibitory effect on AMPA current（P>0.05,n=5）,indicating that the opioid receptor agonist DAMGO had no effect on AMPA current.Conclusion:1.The NMDA current of neurons in the brain area of CFA rACC injected into the soles of rats significantly increased,and the frequency of EPSC increased.2.The agonist DAMGO can activate theμ-opioid receptors in rACC,reduce the NMDA current,and thereby reduce the excitability of neurons. |
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