| Morphine dependence refers to the psychological craving for euphoria caused by chronic morphine,showing symptoms of forced drug seeking behaviors.Depression is caused by long-term stress,“loss of happiness”and anhedonia were known as clinical signs.There seems to be some special connection between these two completely different illnesses.The nucleus accumbens?NAc?,considered as the reward center of the brain.In addition,the NAc is also a key interface for integrating emotions,cognition and behavioral decisions,plays an important role in the addiction and depression.The NAc is devided into two subregions,core and shell,could receive different innervations.In the NAc,there is a 5-hydroxytryptamine?5-HT?pathway projected from 5-HT neurons in the dorsal raphe nucleus?DRN?,which is considered to be important in maintaining basic social activities.It has been reported that morphine can bind to the mu-opioid receptor of gamma-aminobutyric acid?GABA?neurons in DRN,activate 5-HT neurons by inhibiting GABAergic neurons.However,whether 5-HT have effects on the NAc to parpiticate in the regulation of morphine dependent has not been reported.Exploring the regulation of the 5-HT pathway in the DRN-NAc in morphine dependence is of great significance not only for solving the clinical treatment of morphine dependence but also for targeted drug design.At the same time,it is of great significance to understand the functions of the 5-HT system at the level of the neuralcircuit as well as military cognition and applications.Contrary to morphine,one of the core symptoms of depression is loss of pleasure,loss of excitement and feel unhappiness.It is believed that abnormal function of NAc,is one of the reasons for depression.Fluoxetine,a 5-HT reuptake inhibitor,has been used as a clinical drug in the treatment of depression.Its mechanism is to increase the content of 5-HT in the synapse,so that depressed patients feel happy again.There is no definite conclusion about how 5-HT can restore patients'happiness,and its specific effective mechanisms or specific targeting brain areas is still unknown.In addition,because fluoxetine treatment usually takes three weeks to work,the lag of this drug treatment is still a difficult problem in the treatment of depression.Therefore,it is of great significance for basic medical research and clinical application to explore the target brain area of 5-HT in the treatment of depression and find the effective target of5-HT in the treatment of depression.In the first part of this paper,we found that morphine can directly activate 5-HT neurons in DRN by c-Fos immunofluorescence and electrophysiological experiments in mice after acute morphine.In order to verify the role of morphine-activated 5-HT neurons in DRN,we injected adeno-associated virus?AAV?into DRN of wild mice:rAAV2/9-Hsyn-ChR2/eNpHR3.0-mcherry-WPRE-pa;and Fev-cre mice?Insert a cre sequence after the Fev promoter necessary for the 5-HT synthesis process,allowing 5-HT neurons to specifically express cre recombinase?:rAAV2/9-Ef1a-DIO-ChR2/eNpHR3.0-mcherry-WPRE-pa,and implanted optical fibers locally.It was found that photoactivated 5-HT neurons in DRN and induced mice to show real-time conditioned place preference?CPP?,whereas photoinhibited 5-HT neurons in DRN showed real-time conditioned place aversion?CPA?.After chronic morphine injection,photoinhibited 5-HT neurons in DRN,and mice performed no difference in CPP compare to control mice.Therefore,inhibiting 5-HT neurons in DRN alleviates chronic morphine dependence.In addition,intraperitoneal injection of chronic morphine and fluoxetine could induce CPP and mental dependence in mice.It suggests that morphine can activate 5-HT neurons in DRN and promote the release of 5-HT in the whole brain,which may be an important pathway for morphine dependence.In order to explore the target brain regions and functional mechanisms of 5-HT in morphine dependence,we used c-Fos immunofluorescence staining of the forebrain region after acute morphine treatment and found that positive-stanning neurons existed in the NAc shell and mPFC.The NAc shell and mPFC may be the key brain regions for morphine action.At the same time,we found that 5-HT neurons could directly project to the mPFC and NAc shell,and then implanted bilaterally optical fibers into NAc shell and mPFC after local injection of AAV-ChR2/eNpHR3.0 into the DRN of wild-type and Fev-cre mice.We found that photoactivated of the 5-HT axon located in the NAc shell showed real-time CPA,whereas photoinhibition of the 5-HT axon showed real-time CPP only in Fev-cre mice.After chronic morphine injection,photoactivated the axons of 5-HT neurons in NAc shell of wild type and Fev-cre mice,no CPP was shown in mice.Therefore,the release of 5-HT in the NAc shell could alleviate chronic morphine dependence.In contrast,optogenetics activates or inhibits the axons of 5-HT neurons in the mPFC of mice,and there is no difference in real-time CPP between wild-type mice and Fev-cre mice.These results suggest that 5-HT in NAc shell plays a negative role in regulation of morphine dependent.To further understand the mechanism of 5-HT negative regulation on morphine dependencein NAc shell.We analyzed the mechanism of 5-HT effects in NAc shell by electrophysiological method on the field potential and the connectivity?there is connectivity between two or more brain regions when their LFPs were same in a certain frequency band?between the mPFC and NAc shell.We found that photoactivated of 5-HT neurons in DRN resulted in real-time CPP,delta band LFPs was increased and the low gamma frequency band LFPs was decreased in NAc shell,and increased connectivity between NAc shell and mPFC.Photoactivated the axons of 5-HT neurons in NAc shell,the mice showed real-time CPA.Whole frequency band LFPs in NAc shell was decreased,and there is no connection between NAc shell and mPFC.It is suggested that 5-HT is the key factor to relieve morphine dependence by reducing the LFPs of delta,theta and alpha frequencies in NAc shell and weakening the connectivity between NAc shell and mPFC.Summarizing the first part,we found:1.DRN 5-HT neurons directly participate in morphine dependent.2.The release of 5-HT in NAc shell mediated the negative regulation in morphine dependent.3.5-HT decreased the LFPs in NAc shell and reduced the connectivity between NAc shell and mPFC were the reasons for the negative regulation in morphine dependent.Our work in this part provides a new understanding of the function of 5-HT in DRN at the level of neurocircuit,and provides a new targeted pathway and drug pathway for the treatment of morphine dependent.At the same time,the 5-HT released in the NAc shell mediated the escape behavior of mice,which has certain guidance for avoiding the fear,escape and other negative emotions of soldiers in training or combat environment.Therefore,our work has a certain significance in military cognition and military applications.In the second part of this paper,we used the method of in vivo multi-channel electrophysiology and the LFPs as an index to validate the result of PET-CT in depressed rats.The dynamic changes of LFPs in NAc core during chronic fluoxetine treatment were analyzed.We explored how 5-HT work on the NAc core to reverse the depressive behaviors in the level of LFPs and explained the biological significance of LFPs at different frequencies in NAc.First,we used in vivo electrophysiological technology to verify previous results of the PET-CT in depressed rats:NAc core,striatum,prelimbic cortex?PrL?,infralimbic cortex?IL?,anterior nucleus group of dorsal thalamus?ANGDT?,lateral nucleus group of dorsal thalamus?LNGDT?,tegmentum of midbrain?TEG?by the index of LFPs and analysis the NAc core and striatum at the level of neurons.We found that the electrical activity in NAc core was significantly changed at level of neurons and LFPs in depression:4 weeks CUMS mediate the depression by decreae the alpha and beta oscillation in NAc core.Then we analyzed the dynamic changes of LFPs in NAc shell of depressive rats after chronic fluoxetine treatment by electrode implantation.We found that single fluoxetine intraperitoneal injection decreased delta and total frequency band LFPs in NAc core.After 21 days of fluoxetine injection,the alpha and beta frequency band LFPs was significantly increased.Therefore,it is suggested that 5-HT may play a role in the treatment of depression by increasing alpha and beta frequency LFPs in NAc core.Then we designed experiments to explore the biological significance of delta,theta and alpha frequency band LFPs in rat NAc core.We found that the alpha frequency band LFPs in NAc core was increased when the reward stimulation occurred,and was decreased when the nociceptive stimulation occurs.Summary of the second part of this paper:We confirmed that the NAc core plays an important role in occurrence and treatment of depression by electrophysiological verification of the different brain regions of PET-CT in CUMS depressive rats.Chronic5-HT can improved depressive symptoms by increasing the LFPs of alpha and beta frequency band in NAc core.Stimulus types were correlated with the theta band LFPs in NAc core,positive reward stimulus increased and negative injury stimulus decreased.Therefore,our study suggests that NAc core may be a target brain area for 5-HT to improve depression;Chronic 5-HT increases alpha and beta frequency field potentials in NAc core to treat depression;the increase of alpha frequency field potentials in NAc core is related to reward,which may be an effective targeting clues for depression treatment.In conclusion,our second part provides a new perspective for the 5-HT effect to improve the electrophysiological mechanism of depression,giving us new insights into the future clinical treatment of depression by deep brain stimulation,and the development of targeted drugs that can quickly treat depression.This idea,at the same time,can be used as a reference for the pathogenesis,mechanism research and clinical drug design of mental diseases.In order to validate the experimental results above,we used the chronic neuropathic pain rat model as a stimulus factor to build the depression and pain comorbids.Deal with the c-Fos stanning after pain stimulus,we found mPFC and NAc core were related with the mood disorder caused by pain.When rats performed the depressive behaviors in chronic neuropathic pain.In mPFC,the LFPs were decreased and firing rates of interneuron were increased.In NAc core,the delta band oscillation were decreased and beta band oscillation were increased at same time.However,the long-time fluoxetine had no effect on the firing activity of mPFC,but decreased the alpha and increased beta band LFPs in NAc.As a result,chronic 5-HT may improve depressive behaviors in chronic pain by alpha and beta band oscillation.These results revealed that differernt stresses caused the depressive behaviors by different mechanisms,and the fluoxetine,a 5-HT reuptake inhibiter,had different effects to improve the depressive behaviors.Therefore,this part of research has certain guiding significance for the treatment of pain and depression comorbids,or other series of diseases lead to mental complications such as depression. |
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