| ObjectiveGlutamatergic projections from prefrontal cortex（PFc）,amygdala to nucleus accumbens（NAc）regulate the dopamine（DA）release in NAc.However,it is not clear whether these circuits are effective for the reward and motivation of heroin addiction.Our study investigates the effects of metabotropic glutamate receptor 2/3（m Glu R2/3）or glutamate within system or in the circuits from ventromedial prefrontal cortex（vm PFc）,basolateral amygdala（BLA）to the NAc shell on the reward and motivation of heroin-addicted rats,in order to provide theory for early treatment on heroin addiction.MethodsExp.1:First,Rats were trained to self-administration for consecutive 14 days with different doses of heroin（0.0125,0.025,0.05,0.1 mg/injection/kg）.On day 15,rats were injected with m Glu R2/3 agonist LY379268（0,0.1,0.3,1 mg/kg,i.p.）,followed by heroin self-administration testing under fixed ratio 1（FR1）schedule to observe the interaction on heroin reward between LY379268 concentration and heroin dose.Then,other four groups of rats were self-administrated for consecutive 14 days at heroin dose of 0.05 mg/injection/kg,the heroin dose was fixed to 0.05mg/injection/kg in all subsequent experiments.On day 15,rats were injected with LY379268（0,0.1,0.3,1 mg/kg,i.p.）followed by heroin self-administration testing under progressed ratio（PR）schedule,and observed the effect of LY379268 on heroin motivation.Finally,another four groups of rats were trained to heroin self-administration for consecutive 14 days.On day 15,rats were intraperitoneally injected with saline,LY379268（1 mg/kg）,LY341495（1 mg/kg,a m Glu R2/3antagonist）or LY379268+LY341495,and observed the interaction on heroin reward under FR1schedule between LY379268 and LY341495.Exp.2:First,Rats were trained to heroin self-administration for consecutive 14 days.On day15,rats were bilaterally microinjected with different concentrations of LY379268（0,0.3,1 mg/ml）at the volume of 0.5μl into ventral tegmental area（VTA）,NAc core or NAc shell,respectively.Rats were followed by heroin self-administration testing under FR1 schedule to observe the central role of LY379268 on heroin reward.Then,other three groups of rats were trained to heroin self-administration for consecutive 14 days.On day 15,control group was given saline in unilateral vm PFc and contralateral NAc shell.other two groups were given Muscimol+Baclofen（0.06+0.6nmol/L）in unilateral vm PFc or dm PFc,and LY379268（1 mg/ml）in contralateral NAc shell,respectively.The volume per side was 0.5μl,all groups were then received heroin self-administration testing under FR1 schedule.Finally,another two groups of rats were trained to heroin self-administration for consecutive 14 days.On day 15,control group was given saline in unilateral BLA and contralateral NAc shell.BLA-NAc shell group was given Muscimol+Baclofen（0.06+0.6 nmol/L）in unilateral BLA and LY379268（1 mg/ml）in contralateral NAc shell.The volume per side was 0.5μl too,these groups were subsequently received heroin self-administration testing under FR1 schedule.Exp.3:First,rats were injected glutamatergic optogenetical virus or negative control（NC）virus in vm PFc,and trained to heroin self-administration for consecutive 14 days after surgery.On day 15,rats were tested the effects of heroin reward under FR1 procedure by stimulation of glutamatergic neurons in vm PFc.NC group and vm PFc activated group were treated with blue light stimulation in the wavelength of 470 nm,direct current and power of 5 m W.Each stimulation lasting for 1 h and interval for 1 h.Then,other groups were injected glutamatergic optogenetical virus or negative control（NC）virus in BLA,implanted 16 channel electrode in ipsilateral NAc shell,and trained to heroin self-administration for consecutive 14 days after surgery.On day 15,rats were tested the effects of heroin reward under FR1 procedure or heroin motivation under PR procedure by stimulation of glutamatergic neurons in BLA.NC group and BLA group were treated with blue light stimulation in the wavelength of 470 nm,frequency of 25 HZ and power of 5 m W.Each stimulation lasting for 1 h and interval for 1 h.The spike changes in NAc shell terminal were recorded before and after stimulation of glutamatergic neurons in BLA.Exp.4:Rats were injected with rabies virus（RV）in unilateral NAc shell.After 7 d,the rat brain was perfused and the neuron expression in slices was then observed under microscope.ResultsExp.1:First,two-way ANOVA found a significant effect of LY379268 concentration(F（3,106）=3.485,p<0.05)and heroin dose(F（3,106）=3.045,p<0.05),but there was no interaction（p>0.05）.The inhibitory effect of LY379268 on heroin reward was most significant at the heroin does of 0.5 mg/injection/kg.Compared with the control group,heroin infusions of 0.1 and 0.3mg/kg LY379268 group was significantly decreased（p<0.05,respectively）,heroin infusions of 1mg/kg LY379268 group was more significantly decreased（p<0.01）.Then,one-way ANONA found a significant effect of the last ratio completed for final heroin injection(F（3,25）=3.492,p<0.05)and heroin infusions(F（3,25）=5.373,p<0.01).Compared with the control group,the last ratio completed and heroin infusions of 1 mg/kg LY379268 group was significantly reduced（p<0.05,p<0.01,respectively）.Finally,one-way ANONA found a significant effect of the active nose-pokes(F（3,27）=4.084,p<0.05)and heroin infusions(F（3,27）=4.203,p<0.05).Compared with the control group,the active nose-pokes and heroin infusions of LY379268 group was significantly decreased（p<0.05,respectively）.Compared with the LY379268 group,the active nose-pokes and heroin infusions of LY341495+LY379268 group was significantly increased（p<0.05,respectively）.Exp.2:One-way ANONA found no significant effect of the active nose-pokes and heroin infusions between VTA groups（p>0.05,respectively）.There was a significant effect of thebetween NAc core groups,the active nose-pokes and heroin infusions of 1 mg/kg LY379268 group was significantly lower than the control group（p<0.05,respectively）.There was a significant effect of the active nose-pokes(F（2,16）=12.14,p<0.01)and heroin infusions(F（2,16）=11.70,p<0.01)between NAc shell groups.Compared with the control group,the active nose-pokes and heroin infusions of 0.3 mg/kg LY379268 group was significantly decreased（p<0.05,respectively）,the active nose-pokes and heroin infusions of 1 mg/kg LY379268 group was more significantly decreased（p<0.01,respectively）.Then,one-way ANONA found a significant effect of the active nose-pokes(F（2,15）=6.25,p<0.05)and heroin infusions(F（2,15）=5.19,p<0.05)between groups.The active nose-pokes and heroin infusions of vm PFc-NAc shell group was significantly lower than the control group（p<0.05,respectively）.Finally,independent sample T test found no significant effect of the active nose-pokes and heroin infusions between the control group and BLA-NAc shell group（p>0.05,respectively）.Exp.3:One-way ANONA found a significant effect of the active nose-pokes(F（2,15）=5.14,p<0.05),the inactive nose-pokes(F（2,15）=5.32,p<0.05)and heroin infusions(F（2,15）=5.18,p<0.05)between vm PFc groups under FR1 procedure.Compared with the control group,the active nose-pokes and heroin infusions of vm PFc activated group was significantly decreased（p<0.05,respectively）,while the inactive nose-pokes was significantly increased（p<0.05）.Compared with the NC group,the active nose-pokes of vm PFc activated group had a decreasing tendency（p=0.063）.There was no significant effect of the active nose-pokes（p=0.058）and heroin infusions（p>0.05）between BLA groups under FR1 procedure,the active nose-pokes of BLA activated group had a decreasing tendency（p=0.053）when compared with NC group.There was no significant effect of the last ratio completed for final heroin injection and heroin infusions between BLA groups under PR procedure（p>0.05,respectively）.BLA activated group showed obvious neuronal firing at the nerve of NAc shell during light stimulation.Exp.4:BLA and vm PFc had neuron bodies or axons which projected to NAc shell,and the expression location was consistent with the position of those groups treated with LY379268 or optogenetical virus.ConclusionsLY379268 could reduce the reward effect and motivation of heroin addicted rats,and the main central site was located in NAc shell.More evidences showed that m Glu R2/3 activation from vm PFc to NAc shell was involved in the inhibition of heroin reward and motivation.Optogenetic studies found that activation of vm PFc glutamate neurons reduced the active nose-pokes while increased the inactive nose-pokes,might be interfere with extraction reward memory by activation of projection from vm PFc to NAc shell.The activation of m Glu R2/3 and glutamate from BLA to NAc shell had no effect on the reward and motivation of heroin addiction. |
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