The Role Of Muscarinic Type And Its Subtype Receptor In Insula On Rewarding Mechanism Of Morphine

ObjectiveAddiction to drugs is based on pathological changes in brain function produced by repeatedpharmacological insult to the brain circuits that regulate how a person interprets and behaviorallyresponds to motivationally relevant stimuli. Relapses is the important characteristics of addiction, isalso the key problems of the treatment of addiction. Therefore, we focused to observing the effectsof M type and its subtype receptor in insula on morphine reward.MethodsRats were trained to develop conditioned place preference （CPP）. The different images onLED screens on the top of the CPP box serve as discriminative and conditioned stimuli. Rats weretrained with a dose-increasing manner in6cycles （days）.Base line test: A week after surgery, the rats were initially placed in the middle chamber withthe guillotine doors removed for a period of15min to determine baseline preference. During thistime period, spontaneous preference for each compartment was measured.Conditioned trainingThe day after pretesting, the inserts with openings were replaced with solid inserts to fullyseparate the compartments. Place conditioning was conducted using an unbiased andcounterbalanced protocol. Rats were trained with a dose-increasing manner in6training cycles（days）. The initial dose of morphine was5mg·kg^-1 and increased5mg·kg^-1 after finishing every2training cycles. In each training cycle （day）, all of the rats received morphine injection and stayedin CPP box with intended image and bottom for45min in the morning （or afternoon） and receivedsaline injection and stayed in CPP box with the other image and bottom for45min in the afternoon（or morning）.Test of place preferenceIn the next day of last training, The solid inserts were replaced by those with openings, andtime spent in each compartment was monitored for15min. Rats were not exposed to morphineduring this test, the efficacy of CPP establishment was tested. Nearly five hours after testing ofplace preference, we injected drugs either into the insula or injected the insula with saline, and after5minutes they were retested, again during15minutes. Twenty-four hours after testing of place preference, morphine conditioned rat was tested again for place preference, this time withoutcortical injections.ExtinctionThree days following test of place preference, extinction training began. Every three days,the rats had free access to the3compartments for15min. During this time period, spontaneouspreference for each compartment was measured. We have already shown that this extinctionprocedure significantly reduces the expression of morphine CPP.PrimingThe day following extinction, rats received a single day of reinstatement. On this day, all ofthe rats were received an injection of5mg/kg morphine （sc）. After10minutes, we injected drugseither into the insula or injected the insula with saline, and after5minutes they were tested forconditioned place preference.Our present study focuses specifically on the role of the insula in re-appearance of morphineCPP with low dose morphine priming because this region has been supported by evidence ofinsular activity changing with the top–down cognitive modulation of cravings. Therefore, our firstgoal in this research was to investigate the function of muscarinic antagonist receptor scopolaminein the insula cortex. Our second aim was to investigate whether the muscarinic antagonist receptorscopolamine in the insula cortex could inhibit the expression of morphine CPP with low dosemorphine priming. To test this hypothesis, we employed a CPP procedure, it is a useful tool fortesting drugs for anti-craving activity.Results1. The effects of scopolamine in insula for the expression of morphine CPP in ratsCPP scores of Ins-Scopolamine caused significant reductions in morphine CPP respondingwhen compared with vehicle （P <0.05）. These data support that microinjection of scopolamineinto insula inhibit the expression of morphine CPP in rats.2. The function of scopolamine in insula for the expression of morphine CPP with low dosemorphine priming in ratsCPP scores of Ins-Scopolamine caused significant reductions in morphine CPP respondingwhen compared with vehicle （P <0.05）. These data support that microinjection of scopolamineinto insula inhibit the expression of morphine CPP with low dose morphine priming.3. The function of M1-M4antagonist subtype receptor in the insula cortex for the expressionof morphine CPP in ratsCPP scores of M1-pirenzepine caused significant reductions in morphine CPP respondingwhen compared with vehicle （P <0.01）. CPP scores of M4-tropicamide caused significantpromotion in morphine CPP responding when compared with vehicle （P <0.05）.These data support that microinjection of pirenzepine into insula inhibit the expression of morphine CPP inrats. Whereas, microinjection of tropicamide into insula encourage the expression of morphineCPP in rats.4. The effects of M1-M4antagonist subtype receptor in the insula cortex for the expression ofmorphine CPP with low dose morphine priming in ratsCPP scores of M1-pirenzepine caused significant reductions in morphine CPP respondingwhen compared with vehicle （P <0.01）. CPP scores of M2-methoctramine caused significantreductions in morphine CPP responding in recovering phase when compared with vehicle （P <0.05）. These data support that microinjection of pirenzepine into insula inhibit the expression ofmorphine CPP with low dose morphine priming. Microinject methoctramine into insula couldinhibit the expression of morphine CPP with low dose morphine priming in CPP recovering phase.5. The effect of M1、M4agonist subtype receptor in the insula cortex for the expression ofmorphine CPP in ratsCPP scores of M1-MCN-A-343caused significant promotions in morphine CPP respondingwhen compared with vehicle （P <0.05）. CPP scores of M4-LY2033298caused significantreductions in morphine CPP responding when compared with vehicle （P <0.01）.These datasupport that microinjection of MCN-A-343into insula promote the expression of morphine CPP inrats. However, microinjection of LY2033298into insula inhibit the expression of morphine CPPin rats.ConclusionM1subtype receptor in the insula cortex promoted the expression of morphine CPP in rats.M4subtype receptor in the insula cortex inhibited the expression of morphine CPP in rats. M2subtype receptor in the insula cortex could inhibit the expression of morphine CPP with low dosemorphine priming in CPP recovering phase.