岛叶内M型受体及其亚型在吗啡奖赏机制中的作用

发布时间：2018-03-25 08:48

  本文选题：吗啡　切入点：条件性位置偏爱　出处：《宁波大学》2012年硕士论文

【摘要】：目的：药物成瘾是一种以复吸或强迫用药为特征的慢性、反复发作的脑病。海洛因、甲基苯丙胺、可卡因等精神活性物质作用于大脑产生结构和功能性适应的结果从而使机体对毒品和环境产生异常的联系，当再次暴露于伴药线索时可以产生对滥用药物的渴求。复吸是成瘾的重要特征、也是治疗成瘾的关键问题。本课题主要通过向岛叶核团给药来观察M型受体及其亚型的激动剂或拮抗剂在吗啡奖赏机制中的作用，以期加深对成瘾复吸的认识，为防治阿片类物质成瘾戒断后复吸提供理论参考。 方法：建立SD大鼠吗啡条件性位置偏爱（conditioned place preference，CPP）模型，以训练箱体顶部的LED图案和底部的不同结构的地板作为辨别和条件性刺激信号，采用剂量递增法对各组大鼠进行CPP训练。 前测阶段：脑定位手术7d后，将大鼠放入CPP实验箱中，打开中间隔板，让其自由穿梭15min并记录大鼠在CPP箱两侧停留的时间，作为淘汰和分组的依据。 训练阶段：前测后的d2，抽掉中间隔板，条件位置性偏爱实验采用非偏的实验设计方案。训练周期为6d，吗啡的初始剂量为5mg kg~(-1)，每2d递增5mg kg~(-1)，终剂量为15mg kg~(-1)。每天为一个训练周期，，每只大鼠上下午各训练1次。各组大鼠每天上午（或下午）给吗啡，在CPP箱伴药图案侧停留45min；下午（或上午）给生理盐水在CPP箱非伴药图案侧停留45min。若上午注射吗啡，下午则注射生理盐水；次日上午则注射生理盐水，下午注射吗啡，依此类推，交替进行。 测试阶段：CPP测试在条件性训练完成后次日（训练开始后的d7）上午进行。将大鼠放在CPP箱内，抽取中间隔板允许大鼠在CPP箱体三个小室间自由探索15min，并记录大鼠在两侧小室停留的时间。5h后我们向大鼠岛叶区微注射药物或者生理盐水，5min后再次将大鼠放入CPP箱中进行测试15min。24h后对吗啡依赖大鼠再次进行CPP测试，来观察大鼠脑部微注射药物后CPP的恢复情况。 觅药行为的消退阶段：动物建立吗啡CPP行为3d后开始进行消退训练，每隔3d将大鼠放入CPP箱内15min，待测试大鼠在每侧箱体停留的时间基本相同（即CPP得分值基本在零左右）为消退试验标准。 点燃阶段：消退训练结束后的d2，各组大鼠均皮下给予5mg/kg的吗啡，10min后向大鼠岛叶区两侧分别微注射0.5μL药物或生理盐水，5min后进行CPP测试。 CPP作为研究药物依赖性和奖赏效应的行为药理学模型之一，具有设备要求简单，训练周期短等优点，因此在实验中我们选取了CPP作为实验大鼠的训练模型并借用M型受体非选择性抑制剂东莨菪碱来考察M型受体在岛叶中的作用，接下来我们选取了M1受体拮抗剂哌仑西平，M2受体拮抗剂美曲他明，M3受体拮抗剂4-DAMP，M4受体拮抗剂托吡卡胺，M1受体激动剂MCN-A-343，M4受体激动剂LY2033298分别向大鼠岛叶内进行微注射，进而观察各组大鼠CPP的表达情况，从而来研究M型受体亚型在岛叶中的作用，接下来我们通过在点燃阶段向大鼠岛叶区微注射M型受体非选择性抑制剂东莨菪碱，M1受体拮抗剂哌仑西平，M2受体拮抗剂美曲他明，M3受体拮抗剂4-DAMP，M4受体拮抗剂托吡卡胺，来研究岛叶内M型受体及其亚型对低剂量吗啡点燃大鼠CPP的表达的影响。 结果： 1.岛叶内微注射东莨菪碱对吗啡依赖大鼠CPP表达的作用 岛叶-东莨菪碱组CPP的得分值明显低于岛叶-生理盐水组(P 0.05)，结果表明岛叶内微注射东莨菪碱能够显著抑制吗啡依赖大鼠CPP的表达。 2.岛叶内微注射东莨菪碱对低剂量吗啡点燃大鼠CPP表达的作用 东莨菪碱组CPP的得分值明显低于生理盐水组(P 0.05)，结果表明岛叶内微注射东莨菪碱能够显著抑制低剂量吗啡点燃大鼠CPP的表达。 3.岛叶内微注射M1-M4受体拮抗剂后对吗啡依赖大鼠CPP表达的作用 M1-pirenzepine组CPP得分值明显低于生理盐水组(P 0.01)，M4-tropicamide组CPP得分值则显著高于生理盐水组(P 0.05)。结果表明岛叶内微注射M1受体拮抗剂哌仑西平后能显著抑制吗啡依赖大鼠CPP的表达，而岛叶内微注射M4受体拮抗剂托吡卡胺则能促进吗啡依赖大鼠CPP的表达。 4.岛叶内微注射M1-M4受体拮抗剂对低剂量吗啡点燃大鼠CPP表达的作用 M1-pirenzepine组CPP得分值明显低于生理盐水组(P 0.01)，在低剂量吗啡点燃大鼠CPP表达恢复阶段，M2-methoctramin组CPP得分值明显低于生理盐水组(P 0.05)。结果表明岛叶内微注射M1受体拮抗剂哌仑西平后能显著抑制吗啡点燃大鼠CPP的表达。M2受体拮抗剂美曲他明在岛叶内对戒断后低剂量吗啡点燃大鼠CPP的恢复有一定的抑制效果。 5.岛叶内微注射M1、M4受体激动剂对吗啡依赖大鼠CPP表达的作用 M1-MCN-A-343组CPP得分值明显高于生理盐水组(P 0.05)，M4-LY2033298组CPP得分值明显低于生理盐水组(P 0.01)。结果表明岛叶内微注射M1受体激动剂MCN-A-343后能显著促进吗啡依赖大鼠CPP的表达，而M4受体激动剂LY2033298在岛叶内则能抑制吗啡依赖大鼠CPP的表达。 结论：M1型受体在岛叶中可能起促进吗啡依赖大鼠CPP表达的作用，而M4型受体在岛叶中可能对大鼠CPP的表达起抑制作用，M2型受体在岛叶中可能对低剂量吗啡点燃大鼠戒断后巩固记忆的形成产生影响。
[Abstract]:Objective: drug addiction is a relapse or forced medication characterized by chronic, recurrent encephalopathy. Heroin, methamphetamine, cocaine and other psychoactive substances in the brain structure and functional adaptation results so that the body produces abnormal contact of drugs and environment, when exposed to drugs with clues can produce drug abuse craving. Relapse is an important feature of addiction, it is also a key problem in treating addiction. This subject mainly through to the insular nuclei administration to observe M receptor agonists or antagonists in morphine reward mechanism, in order to deepen our understanding of the complex addiction suction, to provide a theoretical reference for the prevention and treatment of opioid addiction and relapse after withdrawal.
Methods: a morphine conditioned place preference (conditioned place preference (CPP)) model was established in SD rats. The LED pattern on the top of the box and floor with different floors at the bottom were used as discriminating and conditioned stimulus signals. CPP training was carried out in each group by dose increasing method.
In the pretest stage: after 7d, the rats were put into the CPP test chamber, and the middle spacer plates were opened, so that they could freely shuttle 15min and record the time for the rats to stay on both sides of the CPP box as the basis for elimination and grouping.
The training phase: pre-test and post D2, removed the middle partition, conditioned place preference experiment using non experimental design. The partial training cycle is 6D, the initial dose of morphine was 5mg kg~ (-1), each 2D increased 5mg (-1) kg~, the final dose of 15mg kg~ (-1) for a day. A training cycle, each rat on the afternoon of the 1 day of training. The rats (morning or afternoon) to morphine, medicine pattern with a CPP box side stay 45min; afternoon (or morning) to saline in the CPP box without drug side pattern for 45min. morning afternoon if the injection of morphine, injection of physiological the next morning, saline; injection of saline, morphine and so on, the afternoon, alternately.
Test phase: CPP test in the conditional after training (training the next day after the start of the morning D7). The rats were placed in the CPP box, the middle partition selected rats were allowed free exploration 15min in the CPP box three chambers, and recorded the rats on both sides of the chamber residence time after.5h I have to insular area rat microinjection of drugs or saline, 5min again after the rats were placed in the CPP box to test 15min.24h on morphine dependent rats CPP test again, to observe the rat brain after injection of CPP micro recovery.
The extinction stage of drug seeking behavior: after the establishment of morphine CPP behavior, 3D began to go out of training, and rats were put into CPP box every 3D. The time to stay in each side box of the rats was basically the same.
At the ignition stage: after the end of the training, rats in each group were given 5mg/kg morphine subcutaneously. After 10min, 0.5 L L or saline were injected on both sides of the insular area respectively, and CPP was tested after 5min.
One of the behavioral model CPP as research on drug dependence and reward effect, has the advantages of simple equipment, short training period, so in the experiment we chose CPP as the training model of experimental rats and M receptor type non selective inhibitor of scopolamine to investigate M receptor in the insula in the role, then we the M1 receptor antagonist of M2 receptor antagonist and Xiping, song he Ming, M3 receptor antagonist 4-DAMP and M4 receptor antagonist tropicamide, M1 receptor agonist MCN-A-343, M4 receptor agonist LY2033298 were micro injection into rats in insula, and observe the expression of CPP in rats, so as to to study the effect of M receptor subtypes in the insula, we lit stage to the insular region of rat microinjection of M receptor type non selective inhibitor of scopolamine, M1 receptor antagonist prazosin and the West Flat, M2 receptor antagonist song he Ming, M3 receptor antagonist 4-DAMP and M4 receptor antagonist tropicamide, to study the insula type M receptor and its subtypes in kindled rats and the expression of CPP effect on the low dose of morphine.
Result锛

本文编号：1662390