【作者】 魏义明；

【导师】 俞昌喜； 陆林； 李素霞；

【作者基本信息】 福建医科大学 ， 药理学， 2010， 博士

【摘要】 成瘾性物质是一类能够影响人类情绪、行为,改变意识状态,并有致依赖作用的化学物质。成瘾性物质滥用既是全球普遍存在的公共卫生问题,又是危害严重的社会问题。复吸是药物依赖状态的一个重要特征。目前认为,导致复吸的主要因素有两个:一是长期存在的精神依赖,导致强迫性觅药和用药行为反复出现;二是药物戒断后长期的睡眠障碍、节律紊乱、躯体症状、负性情绪等稽延性戒断症状。条件位置偏爱(conditioned place preference, CPP)可应用于可卡因等成瘾性物质的精神依赖潜力评价。一些行为学研究发现,药物依赖相关行为,包括自发活动、敏化,条件位置偏爱及自我给药等,都存在昼夜差异,且可能与中脑边缘系统多巴胺能递质系统的昼夜调节有关。然而,有关可卡因条件位置偏爱昼夜差异的脑内分子机制还有待于探索。有研究显示,可卡因依赖者戒断后,虽然不会出现像阿片类物质滥用戒断时明显的躯体症状和自主神经系统紊乱综合症,但是通常会伴有明显的精神症状,如失眠、焦虑、抑郁等,正是这些严重的稽延性症状最终导致可卡因依赖者复吸。但是,可卡因慢性稽延性症状产生的脑内分子机制还有待于研究。最近有研究发现,脑内昼夜节律基因的活动与药物成瘾关系密切,而且这些昼夜节律分子的活性和稳定性受某些激酶,如糖原合成酶激酶3β(GSK-3β)的影响。糖原合酶激酶3β(GSK-3β)普遍存在于机体的各种组织,在神经系统中有高表达,其本质为丝氨酸/苏氨酸蛋白激酶。研究表明GSK-3β参与了体内一系列的信号转导,功能上涉及蛋白合成、细胞增殖、细胞分化、微管形成及神经元凋亡等活动。尤其是近年来发现,非典型的多巴胺D2受体-蛋白激酶B-糖原合成酶激酶3β信号途径(D2-AKT- GSK-3βsignal pathway)与多巴胺样行为的表达及多种精神疾病相关。为此,本研究拟给予大鼠可卡因慢性处理,在观察其条件位置偏爱形成的昼夜差异及戒断后稽延性症状的基础上,探讨这些行为变化与脑内GSK-3β的关系,以期深化理解可卡因精神依赖性以及复吸的神经生物学机制,为其防治提供新的作用途径和新的药物作用靶点。主要内容包括以下两部分:1.可卡因条件位置偏爱的昼夜差异及其与脑内GSK-3β的关系本部分研究包括以下四方面内容:(1)naive大鼠脑内GSK-3β活性的昼夜节律性分析正常SD大鼠在12h/12h昼夜环境(早上5:00开灯,为ZT0;下午17:00关灯,为ZT12)适应7天后。第8天开始,分别在5:00(ZT0),9:00(ZT4),13:00(ZT8),17:00(ZT12),21:00(ZT16),1:00(ZT20)和5:00(ZT24)7个时间点采集大鼠的4个脑区(下丘脑视交叉上核,SCN;前额叶皮层,PFC;中脑腹侧被盖区,VTA和伏隔核,NAc)组织。采用western blot检测不同时间点磷酸化GSK3β/pGSK-3β(GSK-3β磷酸化反应GSK-3β活性下降)的相对水平,并用生物节律余弦分析法和方差分析统计方法,分析GSK-3β活性的昼夜节律性。结果表明,在正常大鼠脑内,SCN、PFC、VTA及NAc中pGSK-3β水平存在着显著的昼夜节律性。(2)可卡因CPP形成的昼夜差异性观察在同样的饲养环境下,分别在ZT4和ZT16时间点,给予大鼠腹腔注射10mg/kg的可卡因,对其进行为期8天的可卡因CPP训练。第9天在相应的时间点测试大鼠可卡因CPP的表达强度(即ZT4点训练,ZT4点测试;ZT16点训练,ZT16点测试),以观察可卡因CPP形成的昼夜差异。结果显示,腹腔注射10 mg/kg的可卡因,可训练大鼠形成可卡因CPP,且有显著的昼夜差异,表现为白天表达显著强于晚上。(3)可卡因CPP测试后大鼠脑区pGSK-3β的检测大鼠可卡因CPP测试结束后,立即在各自时间点(ZT4和ZT16)采集四个脑区(同上)组织,采用western blot检测pGSK-3β水平,并用方差分析比较其差异。结果表明,在SCN区,白天给予可卡因可以显著降低其内pGSK-3β水平,但晚上给予可卡因无显著影响;生理盐水组大鼠pGSK-3β水平,白天高于晚上,而可卡因组pGSK-3β水平无显著昼夜差异。在PFC区,白天或晚上给予可卡因,pGSK-3β水平均无显著变化;生理盐水组大鼠的pGSK-3β水平白天高于晚上,且可卡因组水平依然为白天高于晚上。在VTA区,白天或晚上给予可卡因,pGSK-3β水平均较生理盐水组有显著下降;生理盐水组大鼠的pGSK-3β水平白天高于晚上,且可卡因组水平依然为白天高于晚上。在NAc区,pGSK-3β变化规律类似于SCN区。(4)核团微注射SB216763对大鼠可卡因CPP形成的影响分析上述3部分实验结果,发现大鼠VTA脑区pGSK-3β水平的变化规律与可卡因CPP的昼夜变化最为匹配,故而采取核团微注射方法,在VTA给予GSK-3β活性抑制剂SB216763(1ng/side),观察其对大鼠可卡因CPP形成的影响。结果表明,大鼠VTA核团微注射SB216763后,可卡因CPP在白天和晚上均可形成,但昼夜差异消失。以上结果提示,中脑腹侧被盖区(VTA)GSK-3β活性的昼夜变化参与了可卡因CPP昼夜差异的形成。2.可卡因慢性稽延性症状变化及其与脑内GSK-3β的关系本部分研究包括以下三个阶段:(1)实验分组及可卡因慢性给药本部分实验大鼠在12h/12h昼夜环境(早上8:00开灯,为ZT0;下午20:00关灯,为ZT12)适应7天后,分为可卡因和生理盐水两个大组。对可卡因组大鼠,给予20mg/kg剂量的可卡因连续腹腔注射14天,然后戒断3天、10天和30天,分别命名为d3、d10和d30组;对生理盐水组大鼠,给予同等体积的生理盐水连续腹腔注射14天,命名为saline组。上述四个小组(saline组和d3、d10及d30组)的大鼠再分别设置7个时间点,即:8:00(ZT0),12:00(ZT4),16:00(ZT8),20:00(ZT12),0:00(ZT16),4:00(ZT20)和8:00(ZT24)。每个时间点6只大鼠,共168只。(2)可卡因稽延性症状观察大鼠连续14天腹腔注射可卡因后,戒断3天、10天和30天,分别测定各组大鼠的体重增重;并采用旷场实验、高价十字迷宫和糖水偏爱实验,观察大鼠的自发活动、紧张、焦虑及抑郁样症状等稽延性症状。结果显示,大鼠的体重增重,d3组显著低于saline组,而d10,d30组与saline组比较无显著变化。旷场实验测定的大鼠爬格数(反映大鼠水平运动活性),d3、d10及d30组与saline组比较均显著下降;直立次数(反映大鼠垂直运动活性及探究行为),d3,d10及d30组与saline组大鼠比较无显著差异;粪粒数(反映大鼠紧张状态),d3组显著多于saline组, d10及d30组与saline组无显著差异。高架十字迷宫实验测定的大鼠进入开臂次数及时间比例,d3及d30组与saline组比较无显著差异,d10组显著高于saline组;进入闭臂次数及时间比列,d3及d30组与saline组比较无显著差异,d10组显著低于saline组。大鼠糖水偏爱实验显示,d3组大鼠糖水消费比例显著低于saline组,而d10和d30组与saline组比较无显著性差异。(3)可卡因慢性处理大鼠戒断后脑内pGSK-3β变化的检测上述行为学检测结束后,立即采集各时间点各组大鼠的四个核团(SCN,PFC,VTA及NAc),采用western blot检测不同时间点pGSK-3β的水平,用方差分析和生物节律余弦分析法,分析各脑区pGSK-3β含量及节律的变化。结果表明,在SCN区,saline组大鼠pGSK-3β水平呈现昼夜节律性,可卡因慢性处理后戒断3天,节律性依然存在;而戒断10天和30天,节律性消失;与saline组比较,d3、d10及d30组的大鼠pGSK-3β含量均显著降低。在PFC区,saline组大鼠pGSK-3β水平呈现昼夜节律性,可卡因慢性处理后戒断3天、10天和30天,节律性消失;与saline组比较,d3、d10组大鼠pGSK-3β的含量显著降低;而d30组的显著增加。在VTA区,saline组大鼠pGSK-3β水平呈现昼夜节律性,可卡因慢性处理后戒断3天、10天和30天,节律性消失;与saline组比较,d3、d10及d30组的大鼠pGSK-3β含量均显著增加。在NAC区,saline组大鼠pGSK-3β水平呈现昼夜节律性,可卡因慢性处理后戒断3天,节律依然存在;戒断10天和30天,节律性消失;与saline组比较,d3、d10及d30组的大鼠pGSK-3β含量均显著增加。综合分析以上结果提示,慢性可卡因处理后戒断3天时,大鼠稽延性症状主要表现为体重增重减少、自发活动减少、紧张及轻度抑郁;戒断10天时,主要表现为焦虑;而戒断30天,主要表现自发活动减少。同时,脑内pGSK-3β水平的昼夜节律性,除了SCN和NAc的在戒断3天依然存在外,其余的均消失;而各脑区pGSK-3β的含量,在不同戒断时间均有变化(增加或减少)。由此我们推测,可卡因慢性处理大鼠,戒断不同时间后出现的稽延性症状可能与脑内GSK3β水平及节律性的变化有关,但两者间的确切关系及其机制需待进一步实验证实。综上所述,本研究首次发现:1.正常大鼠脑内(SCN,PFC,VTA及NAc)GSK-3β活性呈现昼夜节律性;且中脑腹侧被盖区(VTA)GSK-3β活性的昼夜变化参与了可卡因CPP昼夜差异的形成。2.可卡因慢性处理后戒断3天、10天和30天,大鼠的稽延性症状各不相同;同时大鼠脑内(SCN,PFC,VTA及NAc)GSK-3β活性及昼夜节律性出现异常,提示两者之间有一定相关性。更多还原

【Abstract】 Addictive substances can affect human emotion , behavior and consciousness, induce dependence after chronic administration. Substance abuse is not only a worldwide public health problem but also a serious social problem. Relapse is an important feature of drug dependence. It is considered currently that there are two main reasons leading to relapse. The first is that long-standing psychological dependence, leading to compulsive drug-seeking behavior and repeated drug use. The second is the persistence of protracted withdrawal symptoms, such as the long-term sleep disorders, rhythm disorders, physical symptoms and negative emotions.Conditioned place preference (CPP) is a behavior pharmacological experimental method to determine drug reward effect of experimental animals, characteristically as simple and short experimental period, and widely used in evaluation of psychological dependence potential. Some behavioral studies had found that drug dependence-related behavior, including spontaneous activity, sensitization, conditioned place preference and self-administration and so on, there is difference between day and night, and may be related to the circadian regulation of the limbic dopaminergic neurotransmitter systems. However, the exact mechanisms need to be further explored.Studies has shown that the sleep quantity and efficiency of heroin abuser is decreased, and after withdrawal , that is often accompanied by some physiological dysfunction such as sleep disorder, diet disorder and emotion disorder so on. Although cocaine withdrawal does not result in the peripheral signs and symptoms of autonomic instability that often seen in opioid withdrawal syndromes, cocaine withdrawal is associated with significant psychiatric symptoms. The severity of cocaine withdrawal symptoms is predictive of treatment dropout and failure to attain abstinence in cocaine-dependent patients participating in outpatient treatment. So studying the mechanisms of cocaine withdrawal may be useful for the prevention of cocaine relapse.Recent studies have found that circadian genes are closely related to drug addiction. And some kinases such as Glycogen synthase kinase 3β(GSK3β), affect the molecular activity and stability of the circadian genes. Glycogen synthase kinase 3βis a ubiquitous serine/threonine protein kinase,which is prevalent in almost all organizations, especially has a high expression in the CNS. Studies have shown that GSK3βinvolved in a series of signal transduction in vivo, has functions related to protein synthesis, cell proliferation, cell differentiation, microtubule formation and neuronal apoptosis and so on. In particular, recent studies have found that an atypical dopamine D2 receptor-protein kinase B-glycogen synthase kinase 3βsignaling pathway (D2R-AKT-GSK-3βsignal pathway), is related to dopamine-like behaviors and a number of mental diseases.So, this study intendes to observe the variation of cocaine CPP and protracted withdrawal symptoms after rats were treated with chronic cocaine. Then try to elucidate the relationship between these behavior and the GSK-3βof brain , in order to further clarify the mechanism involved the relapse of drug-dependence. This study includes the following two parts:1. Diurnal variation of cocaine CPP and its relation to GSK-3βin brain The part of experiment includes the following four elements:(1) Analysis of circadian rhythm of GSK-3βactivity in brainNormal SD rats were adapted in 12h/12h environment (light on at 5:00, defined as ZT0; light off at 17:00, defined as ZT12) for 7 days. The 8th day, we collected the tissue of four brain areas of rats (hypothalamic suprachiasmatic nucleus, SCN; prefrontal cortex, PFC; ventral tegmental area, VTA and the nucleus accumbens, NAc) , respectively in 5:00 (ZT0), 9:00 (ZT4), 13:00 (ZT8), 17:00 (ZT12), 21:00 (ZT16), 1:00 (ZT20) and 5 : 00 (ZT24) . Then, the relative expression levels of phosphorylation GSK3β(pGSK-3β) at different time points were determined by western blot (GSK-3βphosphorylation menas the decreased activity of GSK3β) , and the circadian rhythm of GSK3βactivity was analyzed with biological rhythms cosine analysis and ANOVA.(2) Observation of diurnal variation of cocaine CPPIn the same rearing environment as above, rats were given intraperitoneal injection with cocaine of 10mg/kg, respectively in ZT4 or ZT16, to train cocaine CPP for continuous 8 days. The 9th day, in the corresponding time points (ie ZT4 training, ZT4 test; ZT16 training, ZT16 testing), the expression intensity of rats cocaine CPP was tested in order to observe the circadian differences of cocaine CPP.(3) Detection of pGSK-3βafter cocaine CPP testAfter cocaine CPP test, rats were killed and the tissues of four brain areas (ibid.) were collected immediately, in their respective time points (ZT4 or ZT16). The pGSK-3βexpression was detected by western blot and the difference was analyzed with ANOVA.(4) Effect of nucleus microinjection SB216763 on cocaine CPPAfter analysis of the above three experimental results, we found that the pGSK-3βexpression changes of VTA was most matched with the diurnal variation of cocaine CPP, therefore, SB216763 (inhibitor of GSK-3βactivity, 1ng / side) was microinjected into VTA, to determine the effects of pGSK-3βon the formation of cocaine CPP.The results showed that(1) The expression of phosphoralated GSK-3β(pGSK-3β) in SCN, PFC, VTA and NAC of naive rats has significant circadian rhythm. (2) Rats were trained with cocaine (10mg/kg, ip) and CPP expressed in ZT4 and ZT16 , respectively. The CPP score of ZT4 is higher than that of ZT16.(3) We detected the expression of pGSK-3βin brain after cocaine CPP test, found that(a) In SCN, the expression of pGSK-3βin rats which establish cocaine CPP significantly decreased compared with that of saline, only in ZT4. At the same time, the expression of pGSK-3βwas higher in ZT4 than that of ZT16, only in those rats administrated with saline.(b) In PFC, after treated with cocaine, the pGSK-3βexpression of rats showed no significant differences compared with the saline rats, in spite of ZT4 or ZT16. In the saline rats, the pGSK-3βwas higher in ZT4 than that of ZT16, it showed significance .In rats which treated with cocaine, the pGSK-3βwas significant higher in ZT4 than that of ZT16 also.(c) In VTA, after treated with cocaine, the expression of pGSK-3βof rats was decreased significantly not only in ZT4 but also in ZT16. And, all rats, including that treated with saline or cocaine, the pGSK-3βwas decreased dramatically in ZT16, compare with ZT4.(d) In NAC, the expression of pGSK-3βis similar to that in SCN.(4) After microinjection with SB in VTA, cocaine CPP expression was existed, but it showed no significant differences between ZT4 and ZT16. Those results suggest that the circadian difference of GSK3βactivity in the VTA is critical to the circadian variation of cocaine CPP.2. Protracted withdrawal symptoms of rats after treaed with chronic cocaine and its relation to GSK3βin brain(1) Experimental group and the dosage regimen of chronic cocaine In this part of experiment, rats were adapted in 12h/12h environment (light on at 8:00, defined as ZT0; light off at 20:00, defined as ZT12) for 7 days, and divided into two major groups of cocaine and saline. Rats of group cocaine were i.p. cocaine of 20mg/kg for 14 days, then withdrawal 3 days, 10 days and 30 days, were named as d3, d10 and d30 group respectively; rats of group saline were given the same volume of saline by intraperitoneal injection for 14 days also, named the saline group. All rats (saline group and d3, d10 and d30 group) were divided into seven time points, respectively, at 8:00 (ZT0), 12:00 (ZT4), 16:00 (ZT8), 20 : 00 (ZT12), 0:00 (ZT16), 4:00 (ZT20) and 8:00 (ZT24). There are 6 rats at each time point, so the total is 168.(2) Observation of protracted symptoms of ratsAfter chronic administration , body weight of rats were measured before and after withdrawal to determine weight gain; the spontaneous activity, anxiety and depression-like symptoms of rats were tested by open-field, elevated plus-maze and sucrose preference experiments at different times after cocaine withdrawal to observe the psychiatric symptoms of chronic cocaine.(3) Effect of chronic cocaine administration on the pGSK-3βAfter behavioral test, the relative pGSK-3βexpression of four brain areas (SCN,PFC,VTA and NAc) at different time points were detected by western blot. Then, the intensity and rhythm of pGSK-3βexpression were analyzed by biological rhythm cosine analysis and ANOVA, in order to clarify the relationship between the protracted withdrawal symptoms of cocaine and the changes of brain GSK-3βactivity.We found that(1) Weight gain of rats showed significant differences between group saline and group d3.(2) Open field test (a) The locomotor activity, compared to the group saline, that of group d3, d10 and d30 rats showed significant decrease.(b) The rearing numbers, rats showed no significance between group saline and cocaine.(c) The dejecta numbers, compared to the group saline, which was increased in group d3 and group d10, and decreased in group d30. It showed significant differences between group saline and group d3 only.(3) Elevated plus-maze test(a) Compared with the group saline, the ratio of open arm entry of rats in group d10 was increased, it showed significance. No significance difference showed between the group saline and group d3 or group d30.(b) Compared with the group saline, the ratio of open arm time of rats in group d10 was increased, it showed significance. No significance difference showed between the group saline and group d3 or group d30.(c) Compared with the group saline, the ratio of close arm entry of rats in group d10 were decreased, it showed significance. No significance showed between the group saline and the group d3 or d30.(d) Compared with the group saline, the ratio of close arm time of rats in group d10 were decreased, it showed significance. No significance showed between the group saline and the group d3 or d30.(C) Sucrose preference testThe sucrose consumption ratio of rats in group d3 was decreased, it showed significance. No significance difference showed between the group saline and the group d10 or d30.(3) Changes of pGSK-3βexpression in brain(A) In SCN, it shows a significant circadian rhythm for pGSK-3βexpression in the rats treated with saline and rats abstinent from cocaine for 3 days. No significant circadian rhythm showed in the rats abstinent from cocaine for 10 days and 30 days. After treated with cocaine, the pGSK-3βexpression of all rats showed significant decrease compared with the saline rats.(B) In PFC, it shows a significant circadian rhythm for pGSK-3βexpression in the rats treated with saline. No significant circadian rhythm showed in the rats treated with cocaine [withdrawl 3days, 10days or 30 days]. The pGSK-3βexpression of rats abstinent from cocaine for 3 days and 10 days showed significant decrease compared with the saline rats; but that of rats abstinent from cocaine for 30 days showed significant increase.(C) In VTA, it shows a significant circadian rhythm for pGSK-3βexpression in the rats treated with saline. No significant circadian rhythm showed in the rats treated with cocaine [withdrawl 3days, 10days or 30 days]. After treated with cocaine, the pGSK-3βexpression of all rats showed significant increase compared with the saline rats.(D) In NAc, it shows a significant circadian rhythm for pGSK-3βexpression in the rats treated with saline and rats abstinent from cocaine for 3 days. No significant circadian rhythm showed in the rats abstinent from cocaine for 10 days and 30 days. After treated with cocaine, the pGSK-3βexpression of rats showed significant increase compared with the saline rats.These results suggest that: After treaed with chronic cocaine, when withdrawal for three days, the protracted symptoms of rats include weight gain reduction, lower spontaneous activity, anxiety and depression; withdrawal for 10 days, the protracted symptoms were mainly characterized as anxiety; withdrawal for 30 days, rats showed lower spontaneous activity mainly. Meanwhile, the circadian rhythm of pGSK-3βexpression disappear, except SCN and NAc , when withdrawal for 3 days; and the pGSK-3βexpression of all four brain regions at the different withdrawal times have changed (increase or decrease) . Thus, we speculate that the protracted symptoms of rats treated with chronic cocaine may be related to the expression and rhythm of brain GSK3β, but the precise relationship between the two and the mechanism would need to be further confirmed.In conclusion, this study found first:1. The GSK3βactivity of naive rat brain tissue (SCN, PFC, VTA and NAc) shows circadian rhythmic expression; and the GSK3βactivity of ventral tegmental area (VTA) involves the diurnal variation of cocaine CPP.2. After treaed with chronic cocaine, the activity and circadian rhythm of GSK-3βin rat brain (SCN, PFC, VTA and NAc) are abnormalities, which may be related to the protracted symptoms of chronic cocaine.更多还原