| It is known that presynaptic cannabinoid CB1receptors (CB1Rs), which distributedwidely in nucleus accumbens (NAc), are involved in the natural reward pathway that playan important role in natural drugs (such as opioids or cocaine) addiction and withdrawal.Based on previous findings, in which CB1Rs were found to be involved in the process tomediate striatal GABA synapses one week after the discontinuation of cocaine, we want toknow how the CB1Rs are regulated after withdrawl from morphine (MOR) in NAcneurons of rats. In our study,7days repeated administration of MOR could induce stableMOR–conditioned place preference (CPP). Using electrophysiological experiments, theinhibition effects of CB1Rs agonist WIN55,212–2on spontaneous, miniature and evokedinhibitory postsynaptic currents (sIPSC, mIPSC, eIPSC) were enhanced in both the day3and the day21MOR–treated groups after last injection of MOR. And this effect of MOR exposure could be prevented by AM251, which was perfused before treated with WIN55,212–2. In contrast, the spontaneous, miniature and evoked excitatory postsynaptic currents(sEPSC, mEPSC, eEPSC) and the IPSCs of day35MOR–treated group to WIN55,212–2were unaltered compared with saline–(SAL–) treated rats. Then an experiment wasperformed to measure the retention of the CPP. And we found that the day3and the day21MOR treated–groups had maintained the preference for the MOR–paired compartment,whereas the day35MOR–treated group had extinguished its preference. So we concludedthat the content and/or the capability of CB1Rs were up–regulated and/or enhanced inMOR–treated rats during abstinence of MOR consumption, and the MOR sensitization toCB1Rs could be slowly reversible. The results indicated that regulateing CB1Rs is apromising strategy for the treatment of MOR addiction. The study is composed of fourparts.Part1. The animal model of MOR withdrawal in rats, and determination of theworking concentration of Win55,212–2CPP, which may predict the dependence liability in humans and animals in drugaddiction, is a widely used behavioral measure for the rewarding properties of abuseddrugs. In the present study, we used the CPP paradigm to investigate whether MORadministration produce addictive behavior by testing the rewarding properties at the timepoint of day7after last injection of MOR in rats. Data analysis revealed that repeatedMOR injections could produce significant CPP (P <0.05), whereas the SAL–treated ratsdid not show any significant CPP for either compartment (P>0.05). So, by CPP methods,we could successfully establish the animal model of MOR withdrawal in rats.In the Dose–response relationship assay, we found that0.1μM Win55,212–2was noteffective in both MOR–and SAL–treated rats (P>0.05), while0.3,1,2,4μM Win55,212–2could dose–dependently inhibit the frequencies of sIPSC and the amplitudes ofeEPSC in MOR–and SAL–treated rats. Treatment with Win55,212–2at1μM resulted ina stable effect to rats. Hence, a working concentration of1μM was chosen for allsubsequent studies. Part2. Effects of AM251and Win55,212–2on EPSCs of NAc in SAL–andMOR–treated ratsIn the NAc, stimulation of CB1Rs presynaptically reduces glutamatergic transmission.Thus, to see whether the effects of CB1Rs on drug of addiction and withdrawl wereinvolved glutamate–mediated process, we performed whole–cell recordings to examinesEPSC, and then isolated mEPSC, following the application of tetrodotoxin (TTX), in thepresence of bicuculline. After that, following stimulation around NAc on other brain slices,we examined eEPSC in core region of NAc.Compared with the neurons from SAL–treated rats at the time point of3days,21days and35days, no significant difference were found in frequencies and the amplitudesof sEPSC (P>0.05) and mEPSC (P>0.05), and the amplitudes of eEPSC (P>0.05) afterthe application of the CB1Rs agonist Win55,212–2.Part3. Effects of AM251and Win55,212–2on IPSCs of NAc in SAL–andMOR–treated rats, and the condition of CPP retentionWe measured sIPSC to examine the physiological activity of GABA synapses, and thenisolated mIPSC, following the application of TTX, a selective voltage–dependent sodiumchannel blocker in the NAc of both MOR–and SAL–treated rats, in the presence of APVplus CNQX. After that, following stimulation around NAc on other brain slices, weexamined eIPSC by whole–cell voltage clamp recordings in core region of NAc. All ofthese currents could be blocked by the GABA receptor antagonist, bicuculline.Compared with the neurons from SAL–treated rats at the time point of3days and21days, application of Win55,212–2could significantly reduce the frequencies of sIPSC (P<0.05) and mIPSC (P <0.05), and the amplitudes of eIPSC (P <0.05), which indicatedincreased sensitivity of GABA synapses to cannabinoid receptor stimulation, and theenhanced effect could be prevented by preincubating the slice with the selective antagonistof CB1Rs AM251(P>0.05). But in NAc neurons from day35MOR treated–group,Win55,212–2produced similar effects on the frequencies of sIPSC and mIPSC, and theamplitudes of eIPSC than in the respective controls (P>0.05). Then an experiment was performed to measure the retention of the CPP. And we foundthat the day3and the day21MOR–treated rats had maintained the preference for theMOR–paired compartment. But in day35MOR treated–group, the rewarding effects ofMOR were disappeared.Part4. Effects of Win55,212–2on PPR, and the morphology of MSNs in NAcTo see whether the effects of CB1Rs stimulation on NAc GABA transmission werepresynaptic, PPR analysis was performed to predict the nature of synaptic plasticity. Inboth MOR–and SAL–treated rats, the depressant effect of Win55,212–2on eIPSC, withan interstimulus interval of50ms, was associated with a significant increase in PPR(eIPSC2eIPSC1), as expected for a presynaptic action of MOR (P <0.05for both theMOR–and SAL–groups).The NAc was first identified in coronal slices with reference to the rat brain atlasusing an inverted microscope. By reference to the anterior commissure, we could find theNAc. The MSNs of NAc were found dorsal and medial to the anterior commissure. |
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