| Drug addiction is a chronic and recurrent brain disease with obvious social and psychological characteristics,which is a major medical and social problem.The Chinese government estimated that the addicts of drug in China have exceeded 10 million.Addiction not only causes serious mental and physical harm to the patients,but also induces huge economics(over hundreds of billion yuan per year),society(80%of drug users with crimes)and public health(the spread of AIDS,tuberculosis and hepatitis),which becomes a severe threat to national security and stability.Addiction causes such serious harm mainly because of the susceptibility and high relapse to drug in human(the rate of relapse surpasses 95%within six months after detoxification).Fully understanding the neurobiological mechanism of addiction is the fundamental way to reduce the rate of relapse.While finding effective targets of anti-relapse may help developing ideal medicine and intervention strategies for the treatment of addiction.Clinically,cue is the most critical risk leading to relapse.Neutral stimulus in the environment without rewarding effect are transformed to the cue after repeatedly coupled with drugs in time and space.This pathogenesis involves a series of advanced neuropsychiatric disorders controlled by multiple nervous systems in the brain such as reward,motivation,execution,decision-making,learning,memory and control.The traditional hypothesis is that the reinforcement encoded by the ventral tegmental area(VTA)dopaminergic neurons activates medial prefrontal cortex(mPFC)glutamatergic neurons via the projection from VTA to the mPFC.This process coupled with neutral stimulus,such as sound or light,in time and space results in adaptive changes of glutamatergic neurons in mPFC.On this basis,after the extinction training,the cue can cause the glutamateergic excitement and trigger relapse through the nucleus accumbens(NAc).However,the chemical(pharmic)intervention strategyies developed based on this theory have not effectively solved the problem of cue-induced relapse in the clinic.This is mainly because that it is difficult to obtain accurate functional localization of brain regions,neurons and neural circuits during the addiction.Further,the accurate evidence of information coding and processing between specific neurons in specific brain region and their relationship to behaviors in addiction especially relapse are poorly known.These findings are not only the key points to understand the process of addiction,but also important to discover effective targets for anti-relapse.Admittedly,although these researches are still in the stage of data accumulation,it will undoubtedly be an important breakthrough to effectively reduce the relapse rate of addiction,which is of great significance for comprehensively elucidating the neurobiological mechanism of addiction.Presently,the mesocorticolimbic system is demonstrated the most related neuronal circuits.The system is consisting of dopaminergic(DAergic)neurons in the ventral tegmental area(VTA)and nerve fibers projecting to the nucleus accumbens(NAc)and the medial prefrontal cortex(mPFC).The system constitutes a recognized public pathway for regulating the reward.Various types of addictive substances directly or indirectly increase the concentration of DA in the pathway,producing a strong sense of euphoria,which is accompanied by the creation of learning and memory associated with rewards.This process further induces the emergence of compulsive drug seeking,ultimately leading to addiction.However,in addition to the reward system,drugs simultaneously act on other brain regions,which causes extensive changes in various neurotransmitters and complex neural regulatory networks.How does the mesocorticolimbic dopaminergic system play a specific role in cue-induced relapse?Traditional techniques such as pharmacology,brain damage,and electrophysiology can not be used to specifically dissect the effects of specific neurons and projections in space and time.With the development of research tools in neuroscience,the optogenetics has enabled the understanding of the role of neurons leap from correlation to the causality of function between the specific neural circuits and brain functions.With the optogenetics,recent research has found that specific stimulation of VTA-DAergic neurons can induce positive enhancement;and in the Pavlovian classical model,the specific activation of the VTA-DAergic system can confer the cue motivational value and play the rewarding prediction.At the same time,our studies not only found that the excitation of VTA-DAergic system could encode positive reinforcement,but also for the first time directly proved that the VTA-DAergic neurons dominating the NAc shell,NAc core and IL,but not PL,can encode reinforcement.Therefore,it strongly suggested that the VTA-DAergic system is sufficient to induce addictive behaviors.However,one major question that these hypotheses still cannot answer is whether the VTA-DAergic system can trigger cue-induced relapse and how is it regulated?Relapse occurs after the long-term extinction or withdrawal,during which the central nervous system undergoes extensive neurobiological changes.In other words,although the latest research clarifies that the VTA-DAergic system plays an important role in the initiation of addiction and conferring cue rewarding effect.After a long period of extinction or withdrawal,whether the VTA-DAergic system can still initiate and regulate relapse,and what is the specific pathway and its neurobiological basis?Answering these questions is not only beneficial to the comprehensive and correct understanding of the neurobiological mechanisms of addiction,but also has important scientific significance for discovering potential intervention targets for addiction.Based on the above research,we use specific neuro-manipulation techniques such as optogenetics,chemogenetics and fiber photometry to investigate whether the VTA-DAergic system can directly initiate the cue-induced relapse,and whether this mechanism exists drug addiction?Which VTA-DAergic projections can specifically confer the cue rewarding and motivational value and initiate trigger relapse?The specific results are as follows:1 The regulation of mesocorticolimbic DAergic system on reward-like behaviors and relapse of addictiona)The regulation of VTA DAergic system in reward-like behaviorsDA in the brain play an important role in the process of reward,learning and memory.All kinds of addictive substances can directly or indirectly increase the concentration of DA producing a powerful rewarding effect,which is considered to be the driving factor for the development of addiction.Then,how does the DAergic neurons and various projections in VTA-DAergic system plays a direct role in the reward behavior of addiction?Therefore,this part of the study uses chemical genetics and optogenetic methods to specifically intervene VTA-DAergic system to observe its direct regulation of rewarding behaviors(activity and positive enhancement).(1)The regulation of VTA-DAergic neurons in the motor activityThe hyperactivity is considered to be an important behavioral manifestation.Activated or inhibitory viruses(hM3Dq-YFP or hM4Di-YFP)were injected into the ventral tegmental area(VTA)of DAT-Cre+/-mice by chemogenetics.The virms can be specifically expressed on tyrosine hydroxylase(TH)-positive DAergic neurons.These results suggested that chemogenetic models have been successfully established.For mice expressing hM3Dq-YFP virus,the specific ligand of chemogenetic virus clozapine nitrogen oxide(CNO,1 mg/kg)or vehicle was intraperitoneally injected.The results of behavioral test showed that:Compared to vehicle,CNO-specific activation of VTA-DAergic neurons significantly increased the movement distance of mice(n=6,P<0.01,paired t-test);for mice expressing hM4Di-YFP virus,CNO(1 mg/kg)inhibited VTA-DAergic neurons significantly decreased the movement distance of mice compared to the administration of vehicle(n=5,P<0.01,paired t-test).For mice that were not injected with virus,distance of mice was recorded after intraperitoneal injection of CNO(1 mg/kg)or vehicle.The results showed that CNO had no effect on the activity of the mice compared with vehicle(n=3,P>0.05,t-test).These results suggested that VTA-DAergic neurons directly regulated the motor activity,which is the important basis of reinforcement.(2)The regulation of VTA-DAergic neurons on the reinforcement?.The response curve of excitation of VTA-DAergic neuronsPrevious studies have demonstrated that specifically excitation of VTA-DAergic neurons is able to establish optical intracranial self-stimulation behavior(oICSS).However,only a single stimulation frequency and pulse duration were used,and the conditional cue are accompanied by the training process.Therefore,whether the reinforcement was induced by excitation of VTA-DArgic neurons or the conditional cue itself with incentive value.Therefore,in this reseach,different stimulation frequencies or pulse durations were used to observe the specific changes of reinforcement,thereby clarifying the direct regulation of VTA-DAergic neurons on reinforcement.Using the optogenetics,the activated photosensitive virus ChR2-mCherry was injected into VTA of the DAT-Cre+/"mice.The co-localization of ChR2-mCherry and TH in immunofluorescence experiment suggested that ChR2 was expressed in the TH-positive DAergic neurons in VTA.Establishment of the frequency curve:the laser of 15 ms-pulse duration stimulated VTA-DAegic neurons to observe the oICSS behavior by with multiple frequencies of 1 Hz,5 Hz,10 Hz,20 Hz,25 Hz,50 Hz and 65 Hz respectively.The results showed that the number of active nose-pokes of mice increased significantly with the increasing frequencies(one-way repeated measures analysis of variance(one-way RM ANOVA),frequency main effect P<0.001,n=7);no significant change was observed in inactive nose-pokes(one-way RM ANOVA,frequency main effect P>0.05,n=7).Establishment of the pulse duration curve:the laser at 20 Hz stimulated VTA-DAegic neurons to observe the oICSS behavior by with multiple pulse durations of 2 ms,5 ms,10 ms,15 ms,and 20 ms was used to stimulate the VTA-DA neurons.The results showed that the number of active nose-pokes of mice increased with the length of pulse duration(one-way RM ANOVA,pulse duration maim effect P<0.001,n=5);no significant change was observed in inactive nose-pokes(one-way RM ANOVA,pulse duration major effect P>0.05,n = 5).The results suggested that specific excitation of VTA-DAergic neurons can directly mediate reinforcement.The material basis and receptor mechanisms of reinforcement will be further explored through microdialysis and pharmacological intervention.?.The material basis and receptor mechanisms of reinforcement induced by excitation of VTA-DAergic neuronsIn the immersion animal microdialysis experiment,the excitatory photosensitieve protein ChR2-mCherry was specifically expressed on VTA-DA neurons,and the fiber was embedded in one side of the VTA brain region,and the microdialysis probe was simultaneously embedded in NAc at the same side of VTA.The laser stimulation of 15 ms at 20 Hz was used to specifically excite VTA-DAergic neurons.The cerebrospinal fluid was collected in the NAc at the same side of VTA and detected by high performance liquid meter to measure the DA concentration.The results showed that continuous stimulation of VTA-DAergic neurons within a unit time(one sample every 30 minutes)increased the concentration of DA in the NAc compared with the DA basal level pre-stimulation(n=3,P<0.05,One-way repeated measures analysis of variance).Thus,photostimulation of VTA-DAergic neurons mediate reinforcement by increasing the release of DA.Whether NAc is the main target brain for DA to regulate reinforcement?To answer this question,a 20 Hz 15 ms laser-stimulated VTA-DA neuron was used to establish self-stimulation behavior,and then microinjection of dopamine D3 receptor blocker YQA14(2 ?g/side)was performed on both sides of NAc.To investigate the effect of reinforcement behavior.The results showed that YQA14 injection inhibited the self-stimultion behavior of mice compared with vehicle treatment(Active nose-poke:n=3,P<0.05;inactive nose-poke:n=3,P>0.05).The results suggest that dopamine D3 receptors were involved in reinforcement and that NAc is the major brain region in which DA regulates reinforcement.Dopamine receptors are divided into D1-like receptors and D2-like receptors,and D3 receptors belong to D2-like receptors.Whether the D1-like receptor play a role in reinforcement?The following study will answer this question.The change of frequency curve established by excitation of VTA-DAergic neurons was observed by imtraperitoneal injection of dopamine D1 receptor blocker SCH23390(0.2 mg/kg)or vehicle.The results showed that SCH23390 significantly shifted down the frequency curve by depressing the active nose-pokes(Two-way RM ANOVA:drug main effect P<0.001,frequency main effect P<0.001,interaction P<0.001,n=7),but no effect on inactive nose-pokes(Two-way RM ANOVA:drug main effect P>0.05,frequency main effect P>0.05,interaction P>0.05,n = 7).In summary,through the research above,the regulation effect of VTA-DAergic neurons on the reward-like behaviors(activity and positive enhancement)and the transmitter and receptor mechanisms provide a good experimental basis for the systematic study of the relationship between the mesocorticolimbic DAergic system and cue-induced relapse.Therefore,in the next part,we will focus on the precise contribution and specific regulation of VTA-DAergic system to the cue-induced relapse.(b)The regulaiton of mesocorticolimbic DAergic system on the reinstatement of laser seekingThrough the research above,it is clear that the activation of the VTA-DAergic system can establish reinforcement,it does not mean the establishment of addiction-like behavior.According to the definition of addiction,it can only be identified as addiction if it can be reinstated by related predisposing factors after extinction.Therefore,based on the above experiments,wet further studied the reinstatement of laser seeking behavior(relapse)induced by different stimulating factors,thus determining the regulation of VTA-DAergic system on the relapse of addiction and the specific neural circuits involved in.(1)The reinstatement of laser seeking induced by multiple factors after extinction of oICSS of mesocorticolimbic DAergic neurons?.The reinstatement of laser seeking induced by cue or drugs after extinction of oICSS of mesocorticolhmbic DAergic neuronsMany studies currently suggest that the factors leading to relapse are mainly the cues related to addictive stimuli and the drugs organism addicted in.Therefore,this section we used different kinds of addictive drugs and related cue to study the reinstatement of laser seeking.In the oICSS model,mice were trained to acquire the self-stimulation of VTA-DAergic neurons with the laser stimulation of 15 ms at 20 Hz.After extinction,mice were challenged by cue and drugs(cocaine,methamphetamine or heroin imtraperitoneally injected)respectively.The results were showed as follows:Cue priming:Compared with the average of the last 3 days in extinction,the number of active nose-pokes of mice increased significantly after cue priming(n=6,P<0.05,paired t-test),no significant change was observed in inactive nose-pokes(n=6,P>0.05,paired t-test),which indicated the reinforcement of laser seeking.The result suggested that,in the formation of addiction,activation of the VTA-DAergic system could transfer neural stimulus to the cue by conferring incentive value.Then reinstatement of laser seeking could be induced.Therefore,it is speculated that the VTA-DAergic system may be an important structural and material basis for cue-induced relapse,which is worthy of further study.Drug priming:A general dose that induces relapse in drug addiction,cocaine(10 mg/kg,?.p),methamphetamine(1 mg/kg,i.p.),regression period.Or heroin(1 mg/kg,?.p.),stimulated the experimental animals respectively.We found that,compared with the mean of 3 days of extinction,not only can not cause reinstatement of laser seeking behavior,but also can significantly reduce the active pokes(cocaine:n = 8,P<0.01;heroin:n = 7;P<0.01;methamphetamine:n = 8,P<0.05;paired t test)and the number of inactive nose-poke(cocaine:n = 8,P<0.01;heroin:n = 7;P<0.01;methamphetamine:n = 8,P<0.05;paired t-test).The above results suggest that when a stronger reward effect stimulus is given,a significant substitution effect can be produced to reduce the calendering behavior.Therefore,the above studies showed that VTA-DA neurons directly encode reinforcement and can confer cue reward incentive value,so that the cue can induce reinstatement of laser seeking behavior,which indicating that VTA-DAergic system is a sufficient for inducing addiction-like behavior.The important finding based on cues can significantly induce the relapse,suggesting that the VTA-DAergic system may specifically participate in the initiating and triggering relapse.Therefore,the next section will further study the specific role of VTA-DAergic system for cue-induced reinstatement of laser seeking.?.The reinstatement of laser seeking induced by cue after extinction of oICSS of mesocorticolimbic DAergic projectionsThe study in i.clarified that excitation of the cell bodies of VTA-DAergic neurons could encode reinforcement,whereas the DAergic neurons in VTA projecting to NAc shell,NAc core,IL,and PL may mediate different functions.Then,does the VTA-DAergic neurons confer the cue rewarding and incentive value and trigger cue-induced relapse projecting-specifically?Answering this scientific question will help to find out whether VTA-DA neurons have specific projection pathways in confering cues reward incentive value,initiating and triggering the cue-induced relapse.Using the optogenetics,the activated photosensitive virus ChR2-mCherry was injected into VTA of DAT-Cre+/and specifically expressed in the TH-positive DAergic neurons in VTA.Under the microscope,the red fluorescence of ChR2-mCherry expressed on DAergic projection fibers in the NAc core,NAc.shell,IL and PL could be observed.After ensuring the significant expression of the photosensitive protein,the optic fibers were embedded in the NAc core,NAc shell,IL and PL brain regions respectively.The laser stimulation of 15 ms at 20 Hz and was used to specifically activated the VTA-DAergic neurons projecting to target brain regions(NAc core,NAc shell,IL,and PL)to induce ICSS behavior.After extinction training,mice were challenged with the cue.The results were shown as follows:Activation of the VTA-NAc core,VTA-NAc shell and VTA-IL DAergic projections could induce ICSS behavior,but not the VTA-PL DAergic projection.After extinction,cue priming significantly increased the number of active nose-poke(n=4,P<0.01;n=5,P<0.05;n=4;P<0.05;paired t-test),but not inactive nose-pokes(P>0.05,paired t-test).The above studies suggested that,except for the VTA-PL DAergic projection,activation of other DAergic projections could encode reinforcement and confer the cue rewarding and incentive value,which allowed the cue to induce reinstatement of laser seeking.However,studies have shown that extensive and significant plasticity of the nervous system occurs during extinction.So,even the VTA-DAergic neurons and projections could give the cue to reward incentives,whether the cue still could trigger the relapse after the extiction?If the answer is yes,what about the precise neural circuits?(2)The regulation and specific circuits of mesocorticolimbic DAergic system on cue-induced relapseTo answer the above questions,this part will activation of VTA-DAergic neurons or projections to establish ICSS behavior.After extinction,activation of VTA-DAergic neurons or each projection to induce reinstatement of laser seeking.i.The reinstatement of laser seeking induced by mesocorticolimbic DAergic system after extinction of oICSS of VTA-DAergic neuronsUsing optogenetics,the activated photosensitive virus ChR2-mCherry was injected into VTA of DAT-Cre+/and specifically expressed in the TH-positive DAergic neurons in VTA.Then the optic fibers were embedded in the NAc core,NAc shell,IL and PL brain regions respectively.The laser stimulation of 15 ms at 20 Hz was uesd to train mice to acquire the stable ICSS behavior.After extinction,VTA,NAc core,NAc shell,IL and PL brain regions were stimulated(100 laser stimulation(LS))with laser of 20 Hz 15 ms or 80 Hz 15 ms respectively to activate VTA-DAergic neurons and various DA projections for induction of reinstatement.The results were shown as follows:Compared with the last 3 days in extinction,only activation of the VTA-NAc core DAergic projection significantly increased the number of active nose-poke of mice(n=4,P<0.05,paired t-test),but not active nose-pokes(P>0.05,paired t-test);while activation of VTA-DAergic neurons,VTA-NAc shell DAergic projection,or VTA-ILDAergic projection with 20 Hz 100 LS or 80 Hz 100 LS failed to significantly increase mice's active nose-pokes(n=6,4,4;20 Hz 100 LS:P>0.05;80 Hz 100 LS:P>0.05;paired t-test).While activation of the VTA-PL DAergic projection with 20 Hz 100 LS resulted in a significant reduction in the active nose-pokes compared with the active nose-pokes in extiction(n=4,P<0.05,paired t-test);no significant change in the active nose-pokes was observed when the VTA-PL DAergic projection was activated by 20 Hz 100 LS(P>0.05,paired t-test).So,what is the relationship between the activation of the VTA-NAc core DAergic projection pathway and the cues-induced relapse?Excitation-type photoreceptor virus ChR2-mCherry and inhibitory chemogenetic virus hM4Di-YFP were specifically expressed in TH-positive DA neurons in VTA of DAT-Cre+/-mice,and the stimulation fiber was embedded in VTA and NAc core brain regions.Using a 20 Hz 15 ms laser stimulation to train mice to acquire stable self-stimulation of VTA-DA neurons,followed by extinction until the number of nose pokes was stable.The CNO(0.9 ng/side)was injected into the NAc core to inhibit the VTA-NAc core DA pathway 20 min before the test.It was found that when the vehicle was administered in the same manner,the cue increased the number of active pokes(n=4,P<0.05,paired t-test),but had no effect on the inactive pokes(n=4,P>0.05,Paired t-test),suggesting that the cue induced relapse behavior.However,the number of active pokes was significantly lower than that in the vehicle group when the VTA-NAc core DA projection pathway was inhibited before the cue priming(n=4,P<0.05;paired t-test).The results indicate that inhibition of the VTA-NAc core DA pathway depresses the cue-induced reconstruction of laser seeking.Therefore,it was first discovered that the VTA-NAc core DAergic projection specifically participates in the cue-induced relapse based on the above studies.Is this the result of interaction between the various systems during the initial phase of stimulation of the VTA-DAergic system,or is it possible to activate the VTA-NAc core DAergic projection to initiate relapse?The following researches were further explained.ii.The reinstatement of laser seeking induced by mesocorticolimbic DAergic projections after extinction of oICSS of DAergic projectionsIn this part of the study,the activated photosensitive protein ChR2 was specifically expressed on VTA-DAergic neurons,and then the optic fibers were embedded in NAc core,NAc shell,IL and PL respectively.The laser stimulation of 15 ms at 20 Hz was used to activate each DAergic projection(except VTA-PL)to establish the ICSS behavior.After extinction,each DAergic projection was activated with the laser stimulation of 80 Hz 150 LS were used to induce reinstatement of laser seeking.The results of the experiment showed that only the activation of the VTA-NAc core DAergic projection significantly increased the number of active nose-poke compared to the active nose-pokes in extinction(n=8,P<0.05,paired t-test),but not inactive nose-poke(P>0.05,paired t-test).It is suggested that activation of the VTA-NAc core DAergic projction could initiate the reinstatement of laser seeking.When the VTA-NAc shell and VTA-IL DAergic projctions were activated,there was no significant change in the number of active nose-pokes compared to the extinction(n=5,5,P>0.05;paired t-test).Moreover,after extinction of the ICSS behavior established by activation of VTA-IL DAergic projection,the activation of VTA-NAc core DAergic projection enhanced the number of active nose-poke significantly compared to that in extinction(n=3,P<0.05,paired),and there was no significant change in the active nose-pokes(P>0.05,paired t-test).These results strongly suggested that the VTA-NAc core DAergic projection not only plays an important role in inducing relapse in the initial stage,but also triggers the cue-induced relapse in addiction-like behavior established by other DAergic projections.Therefore,the studies above all can draw the following conclusions:VTA DAergic neurons and projections(except the VTA-PL)can not only encode reinforcement,but also confer the cue reward incentives;After extinction,only the VTA-NAc core DAergic projection is the specific neural pathway that initiates and triggers cue-induced relapse.So,whether this mechanism play an equally important specific role in the cue-induced relapse of drug addiction?Next,in the second part of the study,cocaine self-administration model was used to elaborate it.2.The specific regulation of VTA-NAc core DA projection on cue-induced cocaine relapseThe cocaine self-administration model was used in this part of the study.Firstly,the activity of VTA-DAergic neurons was investigated in cue-induced relapse to determine the direct correlation between them.Then,in the cue-induced cocaine relapse,specific intervention(inhibition and activation)of VTA-DAergic neurons or each projection was performed to clarify the direct regulation of relapse.It was finally determined that the specific VTA-DAergic pathway is necessary for the regulation of cue-induced relapse.The DAT-Cre+/-model animal were used as the objects,and the neural activation indicator,photosensitive protein and chemogenetic inhibitory protein were specifically expressed on DAergic neurons in VTA.These specific neural manipulations combined with mice cocaine self-administration model were carried out in the present study.The specific results were as follows:a)The dynamic activity of VTA-DAergic neurons in cue-induced cocaine relapseThe neuronal activation indicator(GCaMP6m)was firstly expressed specifically on the TH-positive DAergic neurons in the VTA brain region of DAT-Cre+/-mouse,and the recording fiber was embedded in the VTA brain region as well.After confirming the effective expression of the virus,mice were trained with cocaine(0.5 mg/kg,intravenously)to establish self-administration behavior.The mice in one group were given a light as the cue paired with the drug infusion during the training sessions,which is so-called cue-paired group.In the other group,the light was not paired with the drug,that is,the non-cue paired group.After acquisition of the stable self-administration behavior,mice underwent extinction training,and then the two groups of mice were changed by the cue.At the same time,the activity of VTA-DAergic neurons were observed.The results showed that:(1)The number of the number of active nose-poke in the cue-paired group was significantly higher than that in the last 3 days of extinction(n=5,P<0.05,paired t-test),and no significant change was observed in the in active nose-poke(P>0.05,paired t-test).Simultaneously the signal value ?F/F of the fluorescence of the neuronal activation indicator(GCaMP6m)was significantly higher than the extinction(n=5,P<0.05,paired t-test).Besides,the activity of VTA-DAergic neurons maintained at a high level during the period in which the cue appears.(2)For the non-cue paired group,there was no significant change in the active nose-poke of mice compared to the last 3 days of extinction(n=2,P>0.05,paired t-test).The signal value ?F/F of the neuronal activation indicator calmodulin(GCaMP6m)did not change significantly compared to the extinction(n = 2,P>0.05,paired t-test).The results suggested that only the neutral light stimuli can be converted into conditional cues after being paired with the rewarding effect of the drug,thereby being rewarded with incentive value to induce relapse.And the DAergic neurons in VTA could be specifically activated while the relapse was induced by cue.This result strongly suggested that VTA-DAergic neurons directly participated in the cue-induced relapse.Combined with the optogenetic results in the first part,the following methods of chemogenetics and optogenetics were used to inhibit or activate the VTA-DAergic system or VTA-NAc core,NAc shell,IL and PL DAergic projections to intervene of the cue-induced relapse in the cocaine self-administration model,which finally elucidated the specific VTA-DAergic neural projection that initiates and triggers cue-induced relapse.These researches would correspond to the results of the first part.b)The specific regulation of mesocorticolimbic DAergic system on cue-induced cocaine relapse(1)Effects of inhibition of mesocorticolimbic DAergic system on cue-induced cocaine relapseThe inhibitory chemogenetic protein hM4Di-YFP was injected into the brain region of VTA of DAT-Cre+/-mice to specifically express it on TH-positive DA neurons.And specific fluorescent expression of inhibitory proteins was able to be observed on DA neural projection fibers in the brain region,n NAc core,NAc shell,IL and PL.Then,the microinjection cannulas were embedded in NAc core,NAc shell,IL and PL.After ensuring the effective expression of the virns,the mice above were subjected to cocaine self-administration training.After extinction,intraperitoneal injection of clozapine(CLZ,0.1 mg/kg,i.p.)or bilateral infusion of CNO(0.9 ng/side)into NAc core,NAc shell PL or IL were conducted 20 minutes before the cue priming to inhibit VTA-DAergic neurons or VTA-NAc core,VTA-NAc shell,VTA-IL,VTA-PL DAergic projections respectively.The results showed that,after injection of the same dosage of vehicle,the cue could increase the number of active nose-pokes in each group of mice(VTA group:n=4,active nose-poke,P<0.05;NAc core group:n=3,active nose-poke;NAc shell group:n=4,active nose-poke;IL group:n=3,active nose-poke,P<0.001;PL group:n=4,active nose-poke,P<0.01;paired t-test),but had no effect on the inactive nose-pokes(VTA group:n=4,inactive nose-poke,P>0.05;NAc core group:n=3,inactive nose-poke,P>0.05;NAc shell group:n=4,inactive nose-poke,P>0.05;IL group:n=3,inactive nose-poke,P>0.05;PL group:n=4,active nose-poke,P>0.05;paired t-test).These suggested that the cue induced relapse.However,the inhibition of VTA-DAergic neurons or the VTA-NAc core DAergic projection before the cue priming significantly depressed the active nose-pokes of the mice compared to the vehicle group(P<0.05,paired t-test).While inhibition of VTA-NAc shell,VTA-IL or VTA-PL DAergic projections had no significant change in the active nose-poke and inactive nose-poke of mice compared to vehicle(P>0.05,paired t-test).The above results indicated that the VTA-DAergic system was indeed involved in the regulation of cue-induced relapse mainly through the VTA-NAc core DAergic projection.And this pathway played a necessary role in the relapse induced by cue.(2)Effects of excitation of mesocorticolimbic DAergic projections on cue-induced cocaine relapseAfter determining the necessary role of the VTA-NAc core DAergic projection in the cue-induced relapse,VTA-DAergic projections were activated respectively to study the sufficient role in triggering the relapse.The activated photosensitive protein ChR2-mCherry was injected and specifically expressed on VTA-DAergic neurons of DAT-Cre+/-mice,and then the optic fibers were embedded in NAc core,NAc shell,IL and PL respectively.The mice above were subjected to cocaine self-administration training.After ectinction training,the VTA-NAc core,VTA-NAc shell,VTA-IL,and VTA-PL DAergic projections were activated by laser stimulation(100 LS)of 15 ms at 20 Hz to observe the reinstatement of laser seeking,activate respectively.The results showed that only when the VTA-NAc core DAergic projection was activated,the number of active nose-poke of mice enhanced significantly compared to that in the last 3 days of extinction(n=6,P<0.05,paired t-test),but had no effect on the number of inactive nose-poke(P>0.05,paired t-test).However,when 20 Hz,15 ms,100 LS or... |
PDF Full Text Request