| Objective: Morphine, which is widely used for clinic as an analgesicdrug, can relieve the pain of postoperatives and advanced cancers effectively.However, the users are easily going to be morphine dependence which ischaracterized by compulsive drug taking, unsatisfied with present dosage, andthe emergence of withdrawal syndrome after the cessation of drug taking.There are more then90percent addicts who have accomplished thedetoxification come to be relapse, and the relapse mechanism is the emphasisand difficulty. It is worth noting that negative emotional states such as majordepression、bipolar disorder、anxiety et, which are in the course of morphinewithdrawal, are the main factors of relapse after drug abstinence.Present studies suggest that the central non-opioid receptor system maybe an important target for prevention and treatment of addiction, and there areseveral "anti-endogenous opioid peptides" on the regulation of homeostasisafter opioid dependence. Cholecystokinin (CCK), a typical brain-gut peptide,is widely spread in the nervous system and plays an important role in theregulation of varieties of functions as a neurotransmitter or neuro-modulator.Cholecystokinin octopeptide (CCK-8), is the most potent endogenousanti-opioid peptides with the largest content. In our previous studies, we foundthat the exogenous CCK-8could reduce the withdrawal symptom significantlyin morphine withdrawal animals, and be identified a significant inhibitoryeffect of CCK-8on naloxone-precipitated withdrawal-induced conditionedplace aversion (CPA). However, whether CCK-8could inhibit the anxiety-likebehaviors in morphine withdrawl rats, which is good for drug treatment, isunclear.The elevated plus maze (EPM) has been widely used for anxiety-likebehavior on rats and mice or the other rodents at present. The model of morphine dependence and spontaneous morphine withdrawal was set upthrough dose escalation method in our experiment. In order to investigate theeffect of CCK-8on anxiety-like behavior in morphine withdrawal rats and thethe relationship between CCK-8and endogenous opioid system on morphinewithdrawal rats, the anxiety profile was observed on EPM, which is of greatvalue for drug treatment.Methods:1Effect of acute morphine（1,5,10mg/kg，i.p.）treatment on EPManxiety-like behavior in rat was investigated. Percent time spent in, and entriesinto the open arms were used as measure of anxiety.2Effect of acute CCK-8（0.01,0.1,1μg，i.c.v.）treatment on EPManxiety-like behavior in rat was investigated. Percent time spent in, and entriesinto the open arms were used as measure of anxiety.3The time course of anxiety-like behavior in response to morphinewithdrawal in rat: Morphine dependence and spontaneous withdrawal modelwas established by increasing doses manner: injections of morphine forconsecutive5days（10,20,30,40,50mg/kg，i.p.，Bid）), after the inductionof morphine dependence, withdrawal was conducted. During the period ofwithdrawal (day6-12), saline injection (1mL/kg, i.p.) was conducted. Thetime course of anxiety behavior tested by EPM in response to morphinewithdrawal was examined on different times（day1,3,5,7）.4Effect of CCK-8on anxiety profile of morphine-withdrawal rats: Ratsreceived injections of three different doses of CCK-8(0.01,0.1, and1mg,i.c.v.)15min before the EPM testing to observe the effect of exogenousCCK-8on anxiety profile of morphine-withdrawal rats.5Effect of CCK receptor antagonist on CCK-8against anxiety-likebehaviors in morphine withdrawal rat: Rats were injected with CCK1andCCK2receptor antagonist（L-364,718and L-365,260）before the CCK-8treatment, to explore the receptor mechanism of CCK-8on anxiety profile inmorphine withdrawal rats.6The interaction of CCK-8and endogenous opioid system on anxiety profile of morphine-withdrawal rats: Rats were injected with high selectiveμ-opioid receptor antagonist (CTAP,10μg, i.c.v.) and the CCK-8（1μg/rat）15min before EPM testing, to explore the effect of CCK-8and the CTAP onanxiety profile in morphine withdrawal rats. Data was expressed as mean±SEM.The data were subjected to one-way analysis of variance (ANOVA)followed by Bonferroni post hoc test for multiple comparisons with SPSS16.0statistical program, respectively. Values of P<0.05were considered to bestatistically significant.Results:1Acute morphine treatment（1,5mg/kg，i.p.）2h before EPM testingproduced a significant increase in percent open arms time and entries; However, morphine treatment with10mg/kg significantly reduced percentopen arms time and entries in the EPM testing, at the same time the totaldistance traveled by rats was decreased.2There was a significant inhibitory effect of acute CCK-8（0.01,0.1,1μg，i.c.v.）treatment on percent open arms time and entries with no evidentdose-dependent effect in na ve rats. Acute CCK-8treatment has no effect onthe activity in na ve rats.3The chronic morphine withdrawal rats elicited significanttime-dependent anxiety-like behaviors with peak effects on day10(5daysafter induction of morphine dependence).4Administration of exogenous CCK-8（0.01,0.1,1g，i.c.v.） beforeEPM testing on day10produced dose-dependent inhibitory effects onanxiety-like behavior in morphine withdrawal rats. There was a significantincrease in percent open arms time and entries after the treatment of0.1gand1g CCK-8.5The inhibitory effect of CCK-8on percent open arm time and entries inEPM was significantly reduced in L-364,718and CCK-8(1g， i.c.v.)co-treated rats. However, no change was observed in the CCK-8(1g，i.c.v.)plus L-365,260treated group 6Treatment with morphine (5mg/kg, i.p.) before EPM testing effectivelyinhibit the anxiety-like behavior on day10(5days after the induction ofmorphine dependence) in accordance with the effect of CCK-8. CTAP (10g,i.c.v), a selective-opioid receptor antagonist, attenuated the CCK-8’s‘anxiolytic’ effects on morphine withdrawal rats in EPM testing. Moreover,CTAP itself did not change the anxiety-like behavior in EPM testing. Theresults suggest that CCK-8inhibited anxiety-like behaviors in morphine-withdrawal by up-regulating endogenous opioids.Conclusions：Acute morphine treatment attenuates and CCK-8increases anxiety-likebehavior of na ve rats on EPM. But the exogenous CCK-8can inhibit theanxiety-like behaviors significantly in morphine-withdrawal rats byup-regulating endogenous opioids via the CCK1receptor in rats. |
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