The Behavioral-electrophysiological Studies Of Activating The μ-opioid Receptor Of ACC Brain Areas May Alleviate The Affective Pain In Rat

Objective:Nowadays, pain have a significant influence on economy and society, people pay more and more attention on them. Especially the adverse emotional responses which induced by pain affect people’s physical and mental health. So it is necessary and meaningful to study the mechanism of emotional pain and more and more researchers devote themselves to it. A large number of clinical and animal studies have shown, ACC, especially r ACC plays an important role in the emotional pain encoding and adjustment. The opioid receptors also participate in the adjustment of pain. There are lots of opioid receptors on the ACC. So we guess that by activating the opioid receptors on ACC can alleviate the emotional pain. We’ve already had some previous research results of behavioral science and molecular biology. Multi-channel in vivo recording technique is an extracellular recording method, which can monitor the activity of discharge of neurons of free animals and can study the coding mechanism of brain to external events. This study used the multi-channel in vivo recording technique combined with CPA model to observe the change of neuron discharge characteristics of the r ACC μ receptor when they were activated.Methods:CFA-induced the Chronic inflammatory pain models combined with CPA are used to evaluate the emotional pain. We should plant the home-made multi-channel array which combined with bilateral dosing tubes in bilateral anterior cingulate cortex of rats. With the help of micro-injection pump, we can give medication to the ACC. The Neuro Motive and ANY software were used to record and analysis the stay time in the CPA; The cerebus multichannel recording system is used to record the action potential and the local field potential of the ACC neurons of rats and Offline Sorter and Neuroexplorer software are used to sort and analysis the neural signal.The Groups of Experiment are as follows:We can divide the rats into 10 groups according to the left foot subcutaneous injection and the r ACC brain regions to medicine(5-7 of each group):(1) planta NS, r ACC NS;(2)planta CFA, r ACC NS;(3)planta NS, r ACC 1 μg/μl DAMGO;(4)planta NS, r ACC 0.2 μg/μl DAMGO;(5)planta NS, r ACC 0.04 μg/μl DAMGO;(6)planta NS, r ACC 0.01 μg/μl DAMGO;(7) planta CFA, r ACC 1 μg/μl DAMGO;(8) planta CFA, r ACC 0.2 μg/μl DAMGO;(9)planta CFA, r ACC 0.04 μg/μl DAMGO;(10)planta CFA, r ACC 0.01 μg/μl DAMGO.Each group must carry on the experiment for three days:The first day: Pre-conditioning Day, Before the experiment we should put rats in behavior laboratory to adapt for 30 min, then we put one of them in the CPA which is without a baffle in the middle activity freely for 30 min, Recording the time duration of the rat in the two rooms respectively;The following day: Conditioning Day, Before the experiment we should put rats in behavior laboratory to adapt for 30 min, the CPA can divide into two rooms by inserting a baffle in the middle of it. Rats were randomly placed in one of the rooms as unconditioned training room, also known as the “without pain environment”, show pain(-), freely moving for 30 min. After taking out, we give medicine treatment according to different groups. condition matching was conducted after the plantar injection 2h, namely, the rat was put in another room as the conditional training room. also known as the “pain environment”, show pain(+), freely moving for 30 min.The third day: Post-conditioning Day, Before the experiment we should put rats in behavior l aboratory to adapt for 30 min, we put the rat in the CPA which without baffle in middle freely moving for 10 min, Recording the time duration of the rat in the two rooms respectively. Then putting the baffle in the middle of the CPA, we Recorded neuron discharge activity of rats in the CPA rooms respectively using cerebus neural signal recording system, each room recording for 10 min.The CPA score is the time difference of “pain environment” said post-conditioning verse the corresponding said of pre-conditioning.Results:1. HE staining showed: Drugs can be injected into the target brain regions precisely; According to the distance between the medical tube and electrode wire, we know the electrode array are also located in the anterior cingulate cortex.2. Using home-made multi-channel electrode with double metal cannulas, the signal to noise ratio were smaller(SNR<3); Electrical signal could be recorded by using the homemade multi-channel electrode with double quartz capillary without or with polyamide coating, their signals both were excellent(SNR>3). But the bare quartz capillary is harder, fragile and difficult to maintain for a long time in the brain; while the coating one is elastic and easy to insert a plug in it and can maintain a long time.3. The CFA which was injected in the rat plantar can induce the Conditional Position Avoidance response.There has no significant statistic differences when we compared the time of the “pain environment” and the corresponding said in the plantar injected NS group(Post 307.6±29 s VS Pre 306.2±40.27 s, P>0.05, n=6). While there has significant statistic differences when we compared the plantar injected CFA group(Post 210.3±95.36 s VS Pre 323.7±44.82 s, P<0.05, n=6). The score of the avoidance of the groups of plantar injected CFA and NS have significant statistics differences(-113.4±71.13 s VS Pre 1.4±23.02 s, P<0.05, CFA n=10,NS n=6).There has significant statistic differences when we compared the r ACC neuron discharge frequency of the “pain environment” and “without pain environment” in the NS group(Pain(+)2.115(0.4766, 4.163) imp/s VS Pain(-)0.1733(0.0858, 2.289) imp/s, P<0.05, n=49). While there has significant statistic differences when we compared the discharge frequency of the “pain environment” between the group of CFA and NS(2.115(0.4766, 4.163) imp/s VS 0.4533(0.13, 1.415) imp/s, P<0.05, NS n=237, CFA n=49).4. DAMGO injected in r ACC can reverse the CPA response induced by CFAWhen comparing the time of the “pain environment” and the corresponding said of NS+DAMGO group, there has no significant statistic differences(Post 364.6±104.4 s VS Pre 361.3±86.04 s, P>0.05, n=9); The CFA+DAMGO group also has no significant statistic differences(Post 282±117.6 s VS Pre 315.1±66.58 s, P>0.05,n=9); The score of the avoidance of the groups of CFA+DAMGO and NS+DAMGO have no significant statistics differences(-33.13 ± 7137.5s VS 3.322 ± 37.62 s, P>0.05, CFA+DAMGO n=9, NS+DAMGO n=9). There has no significant statistic differences when we compared the r ACC neuron discharge frequency of the “pain environment” and “without pain environment” in the CFA+DAMGO group(Pain(+)0.7058(0.2097, 0.9675) imp/s VS Pain(-) 0.545(0.295, 0.8218) imp/s, P>0.05, n=24); While there has significant statistic differences when we compared the discharge frequency of the “pain environment” between the group of CFA+DAMGO and CFA+NS(0.7058(0.2097, 0.9675) imp/s VS 2.115(0.4766, 4.163) imp/s, P<0.05, CFA+DAMGO n=24, CFA+NS n=49).5. DAMGO has a dose-dependent effect to CPADifferent groups have different CPA response before and after conditional match. there has significant statistic differences in group of CFA+NS(Post 210.3±95.36 s VS Pre 323.7±44.82 s, P<0.05, n=10) and CFA+0.01μg/μl DAMGO(Post 221.9±29.37 s VS Pre 314.4± 38.58 s, P<0.05, n=5); The group of CFA+0.04μg/μl DAMGO/CFA+0.2μg/μl DAMGO/CFA+1μg/μl DAMGO,(Post 207.9±213.3 s VS Pre 362.4±71.67 s, P>0.05, n=6),(Post 272.4±172.9 s VS Pre 353.8±130.3 s, P>0.05, n=6),(Post 282.4±117.6 s VS Pre 315.1±66.85 s, P>0.05, n=9), has no significant statistic differences. The avoidance scores are as follows: There has significant statistic differences in groups. Multiple comparisons shows: There has significant statistic differences in group of CFA+1μg/μl DAMGO VS CFA+NS and CFA+0.2μg/μl DAMGO VS CFA+NS and CFA+0.01μg/μl DAMGO VS CFA+1μg/μl DAMGO. Neurons discharge frequency show that: CFA+NS(Pain(+)2.115(0.4766,4.163)imp/s VS Pain(-)0.1733(0.0858, 2.289) imp/s, P<0.05, n=49) and CFA+0.01μg/μl DAMGO(Pain(+)1.708(0.83, 2.718) imp/s VS Pain(-)0.4942(0.2462, 1.407) imp/s, P<0.05, n=14), has significant statistic differences; CFA+0.04μg/μl DAMGO、CFA+0.2μg/μl DAMGO、CFA+1μg/μl DAMGO(Pain(+)0.4492(0.2363, 2.595) imp/s VS Pain(-)0.5342(0.1971, 2.555) imp/s, P>0.05, n=18),(Pain(+)0.2933(0.1204, 1.15) imp/s VS Pain(-)0.2517(0.1409, 0.7075) imp/s, P>0.05, n=32),(Pain(+)0.7058(0.2097, 0.9675) imp/s VS Pain(-)0.545(0.295, 0.8218) imp/s, P>0.05, n=24), has no significant statistic differences. Neuron discharge frequency of different groups in “pain environment” show: has significant statistic differences in groups. Multiple comparisons shows: There has significant statistic differences in group of CFA+1μg/μl DAMGO VS CFA+NS and CFA+0.2μg/μl DAMGO VS CFA+NS.Conclusion:The behavior and electrophysiological results of the rats are synchronicity, which indicated that the firing characteristics of the neurons have changed when the r ACC μ receptor being activated, DAMGO can reverse the increasing discharge frequency of the neurons which was induced by CFA and play the role of alleviating the affective pain, and this effect have dose-dependency.