Studies On The Role Of PL Area In Medial Prefrontal Cortex In Heroin Addiction And The Underlying Mechanism

Drug abuse can seriously damage the normal physiological functions of the body and lead to a series of social problems.Heroin is one of the widely abused drugs,and research on its mechanism of action can more effectively prevent and treat addictive diseases.Medial prefrontal cortex(m PFC)region plays an important role in drug addiction,drug withdrawal and relapse,and prelimbic cortex(PL)may have a crucial role to drive cocain seeking.Therefore,to uncover the role of PL area in heroin dependence is helpful to explore heroin addiction mechanisms at cortical level and find clues to treat heroin addiction and enable drug withdrawal.This study establish heroin-dependence rat model using traditional conditioned place preference(CPP)system.The movement trajectories of modeling rats were monitored and,the related behavioural parameters were recorded.After damage of PL area with directional lesion techniques,the behavioural change of heroin-addicted rats were re-observed.Further,the impacts of heroin dependence and heroin withdrawal on the EEG waves in the PL area were examined in realtime using a combined techniques of CPP video tracking and wireless telemetry recording methods after coordinated electrode implantation in the PL subregion.The characteristics of EEG wave components and phase-amplitude coupling(PAC)index MI under different states of heroin addiction were analysed relative to control rats.Further,a noncompetitive NMDA receptor antagonist,MK-801,was administered in the PL area of CPP rats,and the rat behavior and EEG waves were re-examined to observe the effects of NMDA receptors blockade on heroin addiction.Additionally,we assessed the expression of all NMDA receptor's subunits in the PL area by Western blotting and q RT-PCR techniques to examine NMDAR expression changes during heroin CPP and withdrawal states relative to control rats.Based on combined results from behavioural tracking,electrophysilogical recording and molecular biology examination,we try to systematically explore the role and mechanism that PL area mediate heroin addiction.Behavioural monitoring results indicated that all heroin dependence rat models were successfully established.Comparison of behavioural data from rats during pre-heroin training,CPP and heroin withdrawal with that from control rats showed that the percentage of time that heroin-withdrawal rats spent in the preferred box(white box)was greatly higher than rats of all other groups(P<0.001),and the percentage of time that CPP rats spent in the preferred box was significantly longer than control rats and rats before heroin induction(P<0.001).These result demonstrated that rats with heroin dependence exhibited evident conditioned place preference.After seven days of heroin withdrawal,rats displayed CPP more evidently.Similar to the time percentage during CPP,the journey and percentage of journey that heroin-withdrawal rats walked in the preferred box was also significantly longer than other groups of rats(p<0.001),indicating a very active drug-seeking behavior during heroin withdrawal.In addition,CPP rats displayed a shortest moving journey(P<0.001)and a slowest moving speed(P<0.001),which indicated that heroin addiction might suppress rat motion behavior.The journey that pre-heroin induction rats rambled in the preferred box was shortest and showed no significant difference with control rats,indicating that the CPP behavior was formed during heroin training in the white box.We also found that heroin-withdrawal rats shuttled more frequently between black and white boxes than other groups of rats(P<0.001),exhibited a serious emotion and hunger for drug reward.After lesion of PL area and heroin administration,the time percentage of rats in the white box was not significantly different from that before lesion and the control group,revealing the important role of the PL area for the formation of conditional position preference.The percentage of movement distance of the control rats in the white box was larger than that before the drug administration(P<0.001),which suggested that the rats formed an adaptation to the preference box,not CPP behavior,and thus moved faster in the preference box.Using wavelet packet algorithm to extract featured frequency waves from EEGs(local field potentials,LFPs),we analysed the detailed changes of LFPs in the PL area during heroin addiction.The percentage of both ? and ?7 waves in the PL area of rats with acute heroin treatment reduced significantly(p<0.05)when compared with that in control rats,while the percentage of ? wave increased significantly(p<0.05)in acute-heroin-treatment rats,and other frequency waves showed no change in acute-heroin-treatment rats relative to controls.The percentage of ?4 wave in CPP rats was lowest and was significantly higher than in control rats(p<0.01)and acute-heroin-treatment rats(p<0.05),whereas the percentage of ? wave increased obviously.These results indicated that all rats with heroin treatment in the white box displayed an evident increase of ? waves and a reduction of ?and ?7 waves,which suggested a possible appearance of cross-frequency coupling.Wavelet packet analysis found that the phase-amplitude coupling(PAC)between ? and?2,?3,?4 and ?5 at varied LFPs frequency ranges(3?5-46?50Hz,80?84Hz;6?8-58?62Hz;6?8-64?68Hz)in acute-heroin-treatment rats reduced significantly relative to control rats(p<0.01).The PAC between ? and ?2,?3,?6 and ?7 at many LFPs frequency ranges(5?7-44?48Hz?48?52Hz?52?56Hz?94?98Hz;6?8-82?86Hz)in CPP rats was evidently increased when compared with that in acute-heroin-treatment rats(p<0.01),but the PAC between ? and ?4(6?8-60?64 Hz)was significantly weakened(p<0.01).Taken together,??? PAC showed a general reduction under the state of heroin treatment,especially the coupling between ? and ?2 and ?3.However,most ???PAC exhibited a reversed increasing after CPP formation.Only ???4 coupling displayed a linear decrease during the process of heroin dependence,suggesting that repeated heroin treatment had a distinct impact on the persistence of ?4 component.After blocking NMDA receptors in the PL area with MK801,the CPP behavior of heroin addiction rats disappeared as shown by a significantly shortened time and time percentage that rats moved in the preferred box.Accompanied with the behavioural change,the percentage of?4 component in the PL area increased significantly,and ? component showed a decreasing trend although the reduction was did not reach a significant level.More specifically,the PAC between ? and ?2,?3,?6 and ?7 at various LFPs frequency ranges(3?5-96?100 Hz?98?102 Hz;4?6-58?62 Hz;5?7-44?48 Hz;6?8-40?44Hz?82?86 Hz)were reversedly decreased(p<0.01),whereas ???4 PAC at the frequency range 6?8-64?68 Hz was reversedly enhanced(p<0.01).Molecular biology studies showed that expression of NR1 subunit of NMDA receptor exhibited no significant difference between control group of rats and rats under CPP status and heroin withdrawal status,suggesting that NR1 is a basic structural component of NMDA receptor,but may not exert regulation function during heroin addiction development.The expression of NR2 subunit showed a different mode between NR2 A,B,C and D: NR2 A expression was stable at different statuses,with a bit higher expression during heroin-induced CPP status but the difference was not significant(p>0.05);NR2B expression level was highest in CPP rats,and exhibited significant difference from that in control rats(p<0.01)and rats under heroin withdrawal(p<0.05).At the same time,q RT-PCR results showed that the m RNA level of NR2 B reached the highest level under the status of heroin-induced CPP,while decreased significantly in the withdrawal status,which indicates that NR2 B may exert an important regulatory role during heroin-induced CPP formation.The expression of NR2 C and NR2 D in the PL area had quite similar paradigm,with a significantly higher expression level in heroin-withdrawal rats than in CPP(p<0.01)and control rats(p<0.01),but no significant difference was found between CPP rats and control ones.In addition,the m RNA content of NR2 C and NR2 D also increased significantly under the heroin withdrawal status.This result indicates that NR2 C and NR2 D may mediate drug-seeking behavior during heroin abstinence.The expression of NR3 A showed a continuously increasing trend from CPP status to withdrawal status during heroin addiction,with the highest expression level during heroin withdrawal period(p<0.001).Further,the NR3 A subunit m RNA level in the withdrawal status was also significantly higher than that in other statuses(p<0.001).The expression of NR3 B under different statuses of heroin dependence,including CPP and drug withdrawal,was no significantly different from that in control rats(p>0.05).In summary,rats display an evident drug-dependence behavioural changes in the process of heroin-induced CPP whereas CPP rats with PL area lesion result in the failure of establishing CPP,which demonstrate that PL area play a key gating role in the formation of conditioned place preference.Further,during the period of CPP and heroin withdrawal,the rhythm of many LFP frequency waves has significant change,especially ?and ?4 waves,which may represent characteristic EEG waves in the process of heroin dependence development.Specifically,the ??? PAC during CPP shows evident difference from the period of acute heroin exposure,with a significantly enhanced ???2,?3,?6,?7PAC and a weakened ???4 PAC,whereas blockade of NMDA receptor in the PL area results in reversed change of these ??? PACs.These results indicate that these ??? PACs are closely related to memory consolidation of heroin addiction and are likely mediated by NMDA receptors.Finally,the expression of NR2 B subunit reach a highest level during CPP status,suggesting that NR2 B may join in the regulation of CPP formation;The expression of NR2 C,NR2D and NR3 A subunits increase continually from CPP period to heroin withdrawal period,indicating their possible role in mediating memory maintainance and relapse of heroin dependence.