| 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.
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