摘要
前言
可卡因成瘾以依赖、戒断困难、易于复吸为主要特征。目前临床上使用的戒毒药因为其本身存在神经毒性和精神行为异常的副作用而未能得到毒品吸食者的广泛接受。寻找安全、有效的戒毒药品是目前戒毒研究领域的当务之急。
大量实验证明褪黑素在药理剂量下发挥多种功效,而且其本身并无明显的耐受性和依赖性。褪黑素改善吗啡依赖大鼠的戒断症状,这一报道为褪黑素作为用于治疗中枢兴奋药依赖的潜在性治疗药物而加以开发利用带来了可能。褪黑素受体在多巴胺环路等脑区广泛分布,且为Gi型G蛋白偶联受体。此外,褪黑素可能作为Ca2+/CaM的拮抗剂结合CaM,并抑制神经系统内含量丰富的CaMKⅡ的激活和自身磷酸化,从而使得CaMKⅡ对神经元的调节功能减弱或丧失。褪黑素通过上述两条或更多的未知途径发挥其负向调节作用。
大量研究表明,可卡因主要作用于中脑边缘多巴胺系统,除影响多巴胺合成限速酶TH的活性外,主要通过阻断多巴胺转运子(DAT)对多巴胺重吸收,进而引起突触的DA堆积以及D1和D2多巴胺受体介导的受体后信号级联反应发挥其中枢兴奋作用。通过观察褪黑素信号通路和可卡因作用的多巴胺信号通路可以发现,AC、cAMP和Ca2+/CaM是两条途径共同的重要分子。
本研究通过采用动物行为学测试CPP(条件性位置偏爱)方法建立大鼠可卡因依赖动物模型,并以褪黑素信号通路和可卡因作用的多巴胺信号通路两条途径共同的重要分子为切入点探讨MT和松果体摘除术对可卡因引燃诱导的CPP强化和可卡因依赖相关蛋白ΔFosB/AGS3/TH在相关脑区表达的作用,为寻找对可卡因依赖有效的药物治疗新靶点提供实验室依据。
方法
1、动物模型建立
(1)大鼠松果体摘除手术及假手术
(2)可卡因依赖大鼠动物模型即CPP的建立、戒断和引燃
2、Western blotting检测褪黑素对可卡因引燃诱导的相关脑区△FosB/AGS3/TH表达的作用
检测盐水组、可卡因组和褪黑素可卡因组三组以及松果体摘除组和假手术组两组大鼠的PFC、NAc、CPu、HIP、AMY和VTA等各脑区内AFosB/AGS3/TH表达。
3、共聚焦激光扫描显微镜技术检测ΔFosB的表达
检测盐水组、可卡因组和褪黑素可卡因组各组大鼠HIP、CPu和NAc部位AFosB的表达。
结果
1、可卡因依赖大鼠模型成功建立、戒断和引燃
(1)可卡因诱导后可卡因组及褪黑素可卡因组CPP均偏爱伴药箱(白箱),可卡因诱导后松果体摘除组的CPP偏爱分数较假手术组的低。
(2)可卡因戒断后各组动物的CPP均恢复到诱导前的生理偏爱。
(3)可卡因引燃后褪黑素可卡因组的CPP偏爱分数显著低于可卡因组。2、Western blotting结果显示褪黑素或松果体摘除可拮抗可卡因引燃对三个靶蛋白的调节
(1)可卡因引燃诱导相关脑区ΔFosB的累积,褪黑素抑制除伏核外相关脑区该分子的表达。松果体摘除抑制可卡因引燃对海马ΔFosB表达的上调。
(2)可卡因引燃下调了腹侧被盖区AGS3表达,上调了AGS3在额前皮质和尾状壳核的表达,褪黑素可抑制此下调和上调作用。
(3)可卡因引燃降低额前皮质和腹侧被盖区TH的表达,增加TH在尾状壳核和杏仁核的表达,褪黑素可抑制此降低和增加作用。松果体摘除抑制可卡因引燃上调杏仁核TH表达的作用。
3、激光共聚焦扫描显微镜技术检测△FosB的表达
可卡因引燃诱导△FosB在HIP.CPu和NAc的高表达,褪黑素拮抗前两个脑区的此诱导作用。
结论
褪黑素拮抗可卡因引燃诱导的CPP强化,褪黑素抑制可卡因引燃诱导的相关脑区内TH.AGS3和△FosB表达。褪黑素拮抗可卡因引燃诱导的CPP强化作用可能是通过拮抗可卡因对相关脑区TH.AGS3和△FosB的诱导实现的。
Introduction
Major features of Cocaine addiction contain long-terming, difficulty to extinct and vulnerability to relapse. Given their side effects that neurotoxicity and abnormal in behaviors, those available medicine for addiction still unpopular at present among people taking in drug. It is urgent priority to look for safe and effective medicine for treatment of addiction.
Results indicated that melatonin possessed multiple functions within pharmacologic dose without tolerance and dependence in itself. The report that melatonin improved morphine induced-abstinent symptom in rats, which brought it possible that development of melatonin as potential medicine for treatment of disorder induced by psychostimulants. Melatonin receptors, coupled to Gαi, widespread in limbic dopamine system. Also, melatonin may antagonize Ca2+/CaM by binding CaM, and inhibit the activation and autophosphorylation of CaMKII, leading to decrease regulation of CaMKII on neuron. Melatonin was reported to execute negative effects by more than both pathways.
It was reported, cocaine affected mainly limbic dopamine system, preventing re-uptake of DAT on dopamine, as well as affecting TH, with dopamine accumulation and receptors mediated-signaling cascade to produce reward. With observing melatonin signaling and cocaine affected signaling pathways, AC, cAMP and Ca2+/CaM were regarded as key molecules in both pathways.
Our study try to investigate the effects of melatonin and pinealectomy on reinstatement of cocaine priming induced-CPP andΔFosB/AGS3/TH expression in related brain regions, by adopting CPP assay and establishing animal model, in view of common molecules in both melatonin pathway and cocaine pathway. And new targets for treating effectively cocaine addiction may be found in present study.
Methods
1. Establishment of animal model
(1) Pinealectomy and Sham-operation in rats
(2) Conditioning, withdrawal, and priming of CPP in animal model of cocaine addiction
2. Effects of melatonin on modulation of cocaine priming inducedΔFosB/AGS3/TH expression is examined by Western blotting
ΔFosB/AGS3/TH expression is examined by western blotting in PFC, NAc, CPu, AMY and VTA from Saline, Cocaine, Melatonin and cocaine, Pinealectomy, Sham-operation.
3.ΔFosB expression is investigated by Confocal microscopy
ΔFosB expression is examined by confocal microscopy in HIP, CPu and NAc from Saline, Cocaine, and Melatonin and cocaine.
Results
1. Animal model of cocaine addiction was successfully established, extinguished, and primed.
(1) Cocaine group and Melatonin-cocaine group preferred for drug-paired compartment after repeated cocaine administration in CPP assay. The preference of Pinealectomy group was lower than Sham-operation group in CPP assay after repeated cocaine administration.
(2) Natural preference was restored after withdrawal followed repeated cocaine administration in each group.
(3) Cocaine priming induced CPP reinstatement in Melatonin-cocaine group, which was significantly lower than that of Cocaine.
2. Results from Western blotting indicated that the regulation of three targets induced by cocaine priming was counteracted by MT or pinealectomy.
(1) Cocaine priming inducedΔFosB accumulation in related brain regions, MT inhibited its expression in most brain regions but NAc. Pinealectomy counteractedΔFosB increase after cocaine priming.
(2) Cocaine priming decreased AGS3 expression in PFC and VTA, and increased AGS3 expression in CPu, both the decreasing and the increasing were inhibited by MT.
(3) Cocaine priming decreased TH expression in PFC and VTA, and increased TH expression in CPu, both the decreasing and the increasing were inhibited by MT. Pinealectomy counteracted cocaine priming induced-TH increase in AMY.
3.ΔFosB expression is investigated by confocal microscppy assay
Results indicated that MT inhibitedΔFosB accumulation in HIP, CPu but NAc, which was induced by cocaine priming.
Conclusions
MT counteracts the reinstatement of CPP induced by cocaine priming. MT inhibited modulation of cocaine priming onΔFosB/AGS3/TH expression. MT counteracts the reinstatement of cocaine priming induced-CPP, which is mediated possibly by TH/AGS3/ΔFosB.
引文
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