| Objective:Pain is the main feature of many diseases,and the lasting impact of chronic pain on patients is far greater than the disease itself.Pain is a comprehensive emotional experience that includes multiple dimensions of feeling,emotion,society,and cognition.Among them,pain makes the patient nervous,anxious,irritable,and even insomnia and depression,which has a great impact on the patient’s quality of life and work efficiency.The mechanism of pain sensation and pain-related emotion caused by persistent nociceptive information uploading to central nervous system are different.Anterior cingulate cortex,as a key brain area in emotional circuit,plays an important role in nociceptive perception and related emotional processing.In particular,the rostral anterior cingulate cortex receives a large number of nociceptive stimulation signals from thalamus and cortex,and participate in the process of producing and regulating pain emotion.As a neurotransmitter in the central system,dopamine plays an important role in decisionmaking,emotion,cognition and other aspects closely related to ACC.Studies have shown that dopamine D1 receptors play a role in alleviating pain-related emotions in other brain regions.It is known that ACC receive projections from dopaminergic neurons in the ventral tegmental area,and there are also dopamine D1 receptors in ACC.It is not clear whether the dopamine D1 receptors can have a similar effect on alleviating negative emotions related to pain in the ACC brain area.Therefore,this topic was used as a scientific question to conduct research.In this project,the persistent inflammatory pain induced by the injection of complete Freund’s adjuvant in the hind paw of the rat was used as an experimental animal model.A small amount of dopamine D1 receptor agonist or antagonist was administered to the ACC brain area of rat.The multi-channel in vivo recording technology was combined with C-CPA test and open field test to explore the role of dopamine D1 receptors in mediating pain-related emotions through the simultaneous observation of behaviors and electricity.Methods:1.Injection of CFA induced persistent inflammatory painCFA was injected into the left hind paws of the experimental animals(0.08 ml),and a blank control group and a normal saline group(0.08 ml)were set up.2.CFA injection induced conditioned place aversionThe injection of CFA on the left hind paws of the rats caused persistent pain.The rats matched the surrounding environment and the pain sensation to produce the pain aversion.After entering the environment again,the rats had obvious aversion behaviors.A blank control group and a normal saline group were set up.3.Multi-channel in vivo recording technologyThe self-made double-sided drug delivery cannula was glued to the recording electrode,and the firing of ACC neurons in each experimental group was observed by multi-channel in vivo recording technology.4.Behaviors testThe rats in each group were administrated with dopamine D1 receptor agonist(10μg)or antagonists(10 μg).Then,one day after administration,the mechanical pain threshold,thermal withdrawal latency,anxiety,motor function and avoidance behavior of rats were tested.5.ImmunofluorescenceIt was confirmed by immunofluorescence test that the dopamine D1 receptor and the GluN1,a subunit of NMDA receptor,were co-expressed in the ACC brain region.Results:1.Injection of CFA on the left hind paws of rats can produce pain-related emotionsWe tested the thermal and mechanical pain behavior of rats in the NAIVE group,the NS group and the CFA group and found that compared with the NAIVE group,the thermal pain and mechanical pain thresholds of the NS group were not statistically different(P>0.05);while the rats in the CFA group were significantly different(P<0.05).It showed that CFA injection can lower the pain threshold and induce hyperalgesia.Next,we measured the C-CPA experiment of rats in three groups to observe the time duration of the rats in the pain environment.Compared with NS group and NAIVE group,CFA group had avoidance behavior to pain environment.The time spent in the pain side pre and post conditioning of the rats in each group was counted.There was no significant difference in the activity time of the rats in each group on 1st day(P>0.05).On the 3rd day,the time of staying on the pain side in the CFA group shortened.There was a statistical difference(P<0.01).It can be seen that the CPA scores of the NS group and NAIVE group were not statistically different(P>0.05);while the CPA scores the CFA group were statistically different(P< 0.001).It indicated that the rats injected with CFA in the left hind paw have aversion behaviors to the pain environment,and the C-CPA model was established.In the OFT,the recording results found that compared with the other groups,the rats in the CFA group had significant changes in their activity trajectories.Rats in the NS group and NAIVE group had no significant difference in movement distance,central activity time and distance percentage(P>0.05);Compared with the blank control group,the CFA group had statistically significant differences in movement distance,the percentage of central residence time,distance in total time and total distance(P<0.001).The results showed that after injection of CFA in the left hind paw,the rats’ open field behavior changed and persistent pain develop negative emotions,which was consistent with the results of CPA test.2.Microinjection of dopamine D1 receptor antagonist SCH-23390 into rat’s ACC can relieve pain-related emotionsRecord the activity trajectories of rats in the NAIVE,CFA+ NS,CFA+ SCH23390 and CFA+ SCH23390 group after CPA test conditioning,and analyze the staying time of the four groups of animals in the pain environment pre and post conditioning.There was no statistical difference in the time before the coupling of the rats in each group(P> 0.05);Compared with the other two groups,the CFA+ NS and CFA+ SKF38393 group had a statistically different time after matching(P<0.0001).The aversion scores of the CFA+NS and CFA+ SKF38393 group were statistically different compared with the other groups(P<0.0001).It showed that microinjection of D1 DR antagonist in ACC can reduce the C-CPA behaviors.From the activity trajectory diagram of each group of rats in the OFT and statistical analysis of the data,it was concluded that the activity distance of the three groups of rats injected with CFA was statistically different(P<0.01);There was no significant difference between CFA + SCH23390 group and blank control group(P > 0.05),but there was statistically difference between CFA+ NS group and CFA+ SKF38393 group(P<0.05).It showed that injection of D1 DR antagonist to ACC can alleviate the anxietylike mood induced by CFA without changing the motor function of rats.3.Injecting dopamine D1 agonist SKF38393 into the rat’s ACC brain area increased negative emotionsThe above experiments showed that D1 DR antagonist could alleviate pain aversion and anxiety in persistent pain model.In order to further verify the role of dopamine D1 receptors in negative emotions,we combined NS+ SCH23390/SKF38393 rats with NAIVE group and NS + NS group rats served as controls.In the CPA test,observed the activity trajectory diagram of each group after coupling and analyzed the activity time of each group pre and post the coupling on the drug delivery side.There was no statistical difference in the activity time of each group before conditioning(P>0.05);while the NS+SKF38393 after training,there were statistical differences in the activity time and CPA scores of rats in the administration side(P<0.05).It showed that the rats injected with D1 DR agonist in ACC had aversion behaviors to the environment of the administration side.The activity trajectory of the open field test was recorded and the movement distance of each group of rats.There was no statistical difference in the movement distance of rats in each group(P>0.05).NS + SKF38393 group compared with other groups,there was a statistical difference in the percentage of active time and distance in center area(P<0.05).It showed that only a small amount of dopamine D1 receptor agonist was injected into the ACC brain area,which did not affect the animal’s motor function but induced anxietylike emotions in the animal.4.The effect of injection of SCH23390 or SKF38393 into the brain region of ACC on neuronal excitabilityStudies have shown that most of the activated neurons in ACC are excitatory neurons in chronic pain.The firing activities of pyramidal neurons in ACC brain area of rats in each experimental group before and after CPA training were recorded,and the firing situation in "pain environment" of rats in each group on the third day was analyzed.In the three groups injected with CFA under the paw,there was no statistical difference in discharge frequency between CFA + SCH23390 group and blank control group(P>0.05).In the three groups of NS injected into the hind paw,the discharge frequency of NS +SKF38393 group was statistically different from the other groups(P<0.05).It was suggested that the normal rats injected SKF38393 in ACC brain area had similar reactions with the rats with persistent inflammatory pain injected with CFA on the hind paw.The D1 DR antagonist reduced the excitability of neurons in the ACC brain region,so it alleviated pain-related emotions.5.Prior injection of SCH23390 into ACC did not alleviate CFA induced hyperalgesiaWe measured the thermal pain threshold and mechanical pain threshold of the rats in each experimental group,and found that there was no statistical difference between the control group and NS injection groups(P>0.05);while CFA injection groups of rats had statistical differences in thermal pain threshold and mechanical pain threshold(P<0.05).It was suggested that the behavioral changes in rats were caused by pain emotion rather than pain feeling.6.Co-expression of dopamine D1 receptor and NMDA receptor in ACCIn order to explore the mechanism of pain related emotion relief by D1 DR antagonists in ACC,we found that a part of ACC neurons D1 DR can be co expressed with GluN1,a subunit of NMDA receptor,which indicated that two kinds of receptors coexist in the same neuron.Conclusion:The dopamine D1 receptors in the ACC brain area of rats can participate in the regulation of pain-related emotions caused by persistent inflammatory pain via affecting the excitability of neurons.The activation of this receptor can up-regulate the excitability of neurons and enhance the response of pain-related emotions. |
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