Studies Of κ-opioid Receptor Activation To Inhibit NMDA Current In Slices Of Anterior Cingulate Cortex Area In Rats Using Patch Clamp Technique

Objective:Pain is one of the most common reasons for patients to seek medical attention.Although it is not immediately life threatening,chronic pain can cause great suffering.The International Pain Association for the study of pain(IASP)latest definition of pain is"pain is a painful experience of related sensory,emotional,cognitive and social dimensions caused by tissue damage or potential tissue damage.The sensory discrimination and emotional experience of pain are conducted by different neural pathways.Numerous studies have shown that ACC is involved in coding painful emotional responses.Ionic glutamate receptors include NMDA receptors and non-NMDA receptors(AMPA and KA receptors),where NMDA receptors are thought to be involved in the processing of the emotional dimension of pain.Opioid receptors are important drug targets for analgesia,addiction and mood disorders.There are three main types of opioid receptors in the ACC brain region:μ-,δ-,andκ-opioid receptors.Previous researches in our laboratory confirmed from behavioral,protein levels,and multichannel electrophysiological records that the injection of CFA on the feet of rats can increase the level of NMDA receptor phosphorylation in the ACC brain region and increase the excitability of neurons,thereby inducing the aversion caused by pain in rats.Activation ofκ-opioid receptors in the ACC brain area can down-regulate the phosphorylation level of NMDA receptor subunits,reduce the excitability of neurons and alleviate the pain and aversion.However,is the increase in excitability of neurons in the ACC brain area caused by the injection of CFA on the soles of the feet caused by increased NMDA current?Does the activation ofκ-opioid receptors can reduce neuronal excitability by inhibiting NMDA currents?There are no firm evidences for this.The purposes of this study were to further demonstrate the relationship between pain and NMDA currents and the relationship betweenκ-opioid receptors and NMDA currents using whole-cell patch-clamp technology.Therefore,the purposes of this study were to use a chronic pain model established by injecting CFA into the soles of rats,and to use brain patch patch-clamp technology to record changes in NMDA currents in ACC brain neurons in rats,and to further explore the excitability of ACC brain neurons and the relationship between NMDA currents changes and the effect of activatingκ-opioid receptors on NMDA currents.Methods:1.Select SD rats(2~3 weeks)and inject 0.08 ml complete Freund’s adjuvant(CFA)on the soles of the feet to establish a model of inflammatory pain.The control group was injected with 0.08 ml of physiological saline(NS).2.Prepare brain slices(250μm)of SD rats containing the ACC region,and use visual patch clamp method to select neurons in good condition to record their NMDA currents.Compare whether the amplitude of large NMDA currents in CFA group rats and control group is different.3.Record the AMPA currents of vertebral neurons in the ACC area,and compare the amplitude of NMDA currents and AMPA currents between the CFA group and the control group.4.Record the spontaneous excitatory post-synaptic current(sEPSP)of vertebral neurons in the ACC region,and compare whether the frequency and amplitude of sEPSP issuance in the CFA group and the control group.5.Add different concentrations(10~-66 mol/L,10~-99 mol/L,10~-1212 mol/L)ofκ-opioid receptor antagonist to brain slice perfusate,and observe their effects on NMDA currents and AMPA currents.6.Add fluorescent dyes to the electrode internal fluid to color the neurons and observe the morphology of the recorded cells.Results:1.When the membrane potential is clamped at+40 mV,the stimulation of the electrode can induce neurons in the ACC brain region of the rat to generate outward currents.Add the AMPA/KA receptor antagonist CNQX(20μmol/L)and the GABA receptor antagonist Bicuculline(100μmol/L)to the perfusate,the currents are partially reduced,and then add the NMDA receptor antagonist AP-5(100μmol/L)to the perfusate.The current is all suppressed,which proves that the current is the NMDA current.If the NMDA receptor antagonist AP-5(100μmol/L)and the GABA receptor antagonist Bicuculline(100μmol/L)are added to the perfusate,the current is mostly reduced;then the AMPA/KA receptor antagonist is added to the perfusate CNQX(20μmol/L),it can be seen that the current is all suppressed,indicating that the current suppressed by CNQX is the AMPA current.2.The membrane potential was clamped at+40 mV,and the AMPA/KA receptor antagonist CNQX(20μmol/L)and the GABA receptor antagonist Bicuculline(100μmol/L)were added to the perfusate to record the currents induced by the stimulation electrode in rats’ACC.NMDA currents of neurons in the ACC could be recorded.Compared with the rats in the control group,the amplitude of NMDA currents in the neurons of the ACC brain area of the rats injected with CFA in the sole of the foot increased significantly(P<0.05,n=5).3.Membrane potential was clamped at+40 mV,and NMDA receptor antagonist AP-5(100μmol/L)and GABA receptor antagonist Bicuculline(100μmol/L)were added to the perfusate to record the AMPA currents induced by stimulation electrodes in the ACC brain.Compared with the rats in the control group,the amplitude of AMPA currents in the neurons of the ACC brain area was not significantly different with the rats injected with CFA in the sole of the feet(P>0.05,n=5).4.Spontaneous excitatory post-synaptic current(sEPSP)was recorded in Gap-free mode.Compared with control group rats,the frequency and amplitude of sEPSP release in ACC brain neurons in CFA group rats increased significantly.5.Selected the cells in the brain slices of the CFA group,clamp the membrane potential to+40 mV,and added the AMPA/KA receptor antagonist CNQX(20μmol/L)and the GABA receptor antagonist Bicuculline(100μmol/L)to the perfusate.The NMDA currents of rat ACC brain neurons induced by stimulation electrodes can be recorded.Different concentrations ofκ-opioid receptor agonist Dynarphin A were added to the perfusate.It was found that 10~-1212 mol/L Dynarphin A had no significant effect on NMDA current;10~-99 mol/L Dynarphin A could significantly reduce NMDA currents in CFA rats.(P<0.05,n=5),the effect of 10~-66 mol/L Dynanorphin A is more significant(P<0.01,n=5),indicating that with the increase ofκ-opioid receptor agonist concentration,the degree of inhibition of NMDA currents also gradually increased.6.Selected the cells in the brain slices of CFA group rats,clamp the membrane potential to+40 mV,and added the NMDA receptor antagonist AP-5(100μmol/L)and the GABA receptor antagonist Bicuculline(100μmol/L)to the perfusate.AMPA currents in rat ACC brain neurons induced by stimulation electrodes could be recorded.The effect of adding different concentrations ofκ-opioid receptor agonist Dynorphin A to the perfusion fluid on AMPA currents in CFA group rats was not statistically different(P>0.05,n=5),which proved thatκ-opioid receptor had no significant effect on AMPA currents.7.After fluorescent staining of brain slices in the ACC area of rats,it can be clearly observed that the cell bodies,axons and dendrites recorded in the experiment show green fluorescence.Conclusion:The injection of CFA in the feet of rats caused a significant increase in the amplitude of NMDA receptors(not AMPA receptors)-mediated currents in the ACC brain area,which is the reason for increasing the excitability of these neurons;and activation ofκ-opioid receptors reduces the amplitude of NMDA currents to reduce the excitability of neurons.