【作者】 孙吉叶；

【导师】 吴春福；

【作者基本信息】 沈阳药科大学 ， 药理学， 2011， 博士

【摘要】 吗啡(morphine)对中枢神经系统产生广泛而复杂的作用。抗坏血酸(ascorbic acid, AA)具有抗氧化以及酶的辅助因子等多种作用,并且在中枢神经系统中发挥着重要的神经调质作用。调节AA的释放可能是成瘾性药物的共同特征。我们研究发现大鼠伏隔核(nucleus accumbens, NAc)透析给予吗啡与全身给予吗啡时引起NAc内AA释放的结果不同,提示不同方式给予吗啡引起NAc中AA的释放存在不同的调节机制。基于此,本文从神经生化、行为学和分子生物学等多个水平研究吗啡引起NAc内AA释放的作用机制,以期为吗啡中枢作用机制研究提供理论依据。首先采用脑微透析结合高效液相色谱-电化学检测和高效液相色谱-荧光检测方法考察了局部NAc透析给予吗啡引起NAc内AA、谷氨酸(glutamate, Glu)、γ-氨基丁酸(γ-aminubutiric acid, GABA)释放的影响。随之考察阿片受体拮抗剂纳洛酮对吗啡作用的影响。结果表明,吗啡(100μM,1mM)能够剂量依赖性地增加NAc内AA和GABA的释放,降低Glu的释放。给予纳洛酮能够显著抑制吗啡引起NAc内AA释放的增加,同时能够抑制吗啡引起的GABA释放的升高,但不影响吗啡引起的Glu的降低。提示吗啡至少是部分通过作用于阿片受体,尤其是NAc内p受体,而产生的对AA和GABA释放的一系列作用。进一步采用海人藻酸(kainic acid, KA)损毁NAc考察其对吗啡作用的影响。结果表明,NAc损毁抑制了吗啡引起NAc内AA和GABA释放的增加,但不影响吗啡引起的Glu释放降低。提示,KA损毁可能导致了NAc内主要的GABA神经元的缺失从而产生一系列效应。为了验证上述推测,又采用NAc局部注射GABAA受体激动剂muscimol考察其对NAc损毁后吗啡作用的影响。结果表明,muscimol能够逆转KA损毁NAc引起的吗啡对AA和GABA释放的抑制作用,提示NAc内GABA能系统在一定程度上参与了吗啡引起的NAc内AA的释放。此外,采用KA损毁NAc的方法,考察其在影响吗啡引起生化水平改变的同时,是否会对吗啡引起的行为学改变产生影响。结果表明,KA损毁NAc能够增加单次吗啡引起的大鼠水平和垂直运动次数；能够部分地阻断大鼠急性成瘾时的戒断症状；强化吗啡对小鼠的镇痛作用；不影响吗啡诱导的小鼠行为敏化的形成,但能阻断吗啡诱导的小鼠行为敏化表达。提示NAc作为中脑边缘重要核团,在吗啡引起的相关行为学中发挥着重要作用。NAc接受来自其他脑区的神经投射。本文进一步考察了前额叶皮层Glu能系统和腹侧被盖区(ventral tegmental area, VTA)对伏隔核的神经投射对吗啡引起AA释放的影响。结果表明,前额叶皮层-NAc的Glu能神经投射降低NAc内的Glu基础释放,不影响GABA基础释放,进一步确证了前额叶皮层-NAc的谷氨酸能神经投射不是参与吗啡作用的主要因素。VTA内注射GABAA受体拮抗剂bicuculine和激动剂muscimol影响NAc中AA的基础释放,muscimol不影响吗啡的作用,但bicuculine能够拮抗吗啡引起的NAc中AA和GABA释放的增加；同时,VTA内注射bicuculine显著升高多巴胺(dopamine, DA)的基础释放,降低NAc中GABA的基础释放,而muscimol作用与之相反。提示VTA到NAc的神经投射可能参与了吗啡引起的NAc中AA释放过程。已知脑内AA主要是通过Na+依赖的AA转运体(SVCT2)转运入脑和神经元从而发挥作用的,并且能够逆浓度梯度在细胞内聚集AA。本文进一步采用RT-PCR、Western blot的方法,考察了上述研究过程中吗啡对脑内AA转运体表达的影响。结果表明,全身给予吗啡能够剂量依赖性地增加纹状体中SVCT2 mRNA和蛋白的表达；NAc透析给予吗啡能够增加NAc内SVCT2 mRNA和蛋白的表达；NAc给予纳洛酮和VTA给予bicuculine能够拮抗吗啡引起的NAc中SVCT2 mRNA和蛋白表达的增加。提示,在吗啡通过作用于受体及神经系统等引起NAc内AA水平改变的过程中,SVCT2可能发挥着重要作用。综上,NAc内的GABA能神经系统参与了吗啡引起的NAc中AA的释放；KA损毁NAc不但改变吗啡的神经生化水平作用,还影响吗啡镇痛和行为敏化的行为学作用；前额叶皮层-NAc的Glu能神经通路在吗啡引起NAc内AA释放过程中可能不发挥主要作用,而VTA-NAc的DA能神经通路参与了吗啡的作用。在吗啡引起AA释放的同时,对SVCT2的表达也产生影响。上述研究不仅有助于深入了解吗啡对NAc内AA释放的调节机理,同时也为进一步了解吗啡中枢作用提供依据。更多还原

【Abstract】 Administrated with morphine can produce many complex neurobiological effects in the central nervous system (CNS). In recent years, increasing evidence shows that ascorbic acid (AA) is a potent neuromodulator in the central nervous system to modulate some nervous activities. Drugs, such as morphine, induce AA release in the nucleus accumbens (NAc) after acute administration, suggesting that AA release in NAc might be a common feature in response to addictive drugs. Previous studies have shown the different effects of morphine on AA level following local infusion. The phenomenon indicates the existence of different regulating mechanisms of morphine in this nuclear. To further understand the neuromechanisms underlying morphine, in the present study we investigated the pharmacological, neuroanatomical, neurobiochemical and molecular alterations in the NAc following morphine administration.Firstly, the roles of the NAc glutamatergic pathways in local infusion of morphine-induced AA, glutamate (Glu) andγ-aminobutyric acid (GABA) release were studied by using microdialysis coupled to high performance liquid chromatography (HPLC) with electrochemical detection and HPLC with fluorescent detection. The results show that morphine (100μM,1 mM) dose-dependently increases the levels of AA and GABA, and decreases the level of Glu in the NAc. However, the effects of morphine on AA and GABA release could be reversed by naloxone. The results indicate that opioid receptor was involved in morphine-induced AA release in the NAc.To further investigate the effects of morphine, the method of NAc lesioned by kainic acid (KA) was used in this study. KA lesions eliminated the effects of morphine on the increasing level of AA and GABA in the NAc, but not the Glu. KA is thought to destroy the GABAergic neuronal cells. The muscimol (100 ng) was administrated to the NAc to prove the hypothesis. It was shown that muscimol could reverse the effect of KA lesion on morphine-induced AA and GABA release, indicating that GABAergic system in the NAc is involved in the morphine-induced AA release.In the present study, we also investigated the effects of KA lesion on morphine induced behavioral sensitivity. In the test of locomotor activity, lesions of NAc affected the locomotor activity of horizontal movement induced by morphine. Meanwhile, NAc lesions enhanced the pain threshold induced by morphine (5 mg/kg). In the behavioral sensitization tests, lesions of NAc blocked the induction of behavioral sensitization by repeated morphine, but not the hyperactivity by acute morphine and the induction of behavioral sensitization.With the consideration of the neuron projection in the limbic midbrain, we also examine the effects of medial prefrontal cortex (mPFC) and ventral tegmental area (VTA)-projection to the NAc on the AA release induced by morphine. Undercutting the mPFC could cut down the glutamatergic cortico-accumbens pathways. While the results showed it did not affect morphine-induced AA release in the NAc. Bicuculline (150 ng), the GABAA receptor antagonist, could inactivate GABAA receptors located on GABA interneurons, inhibit their activity and then, in turn, disinhibit the activity of DA cells, and increase the dopamine (DA) release in the NAc, which finally was related to the effects of blocking the AA release induced by morphine in the NAc. It was indicated that the effect of morphine on AA release in the NAc is partically regulated by the GABAA receptor-mediated action of DA afferents from the VTA.AA is transported into the brain and neurons via the sodium-dependent vitamin C transporter 2 (SVCT2), which causes accumulation of AA within cells against the concentration gradient. In this study, we also investigate the effects of morphine on the mRNA and protein expression of SVCT2. The results show that morphine (i.p.) significantly increase the expression of SVCT2 in the striatum, but not the NAc, while, morphine (1mM) significantly increased the expression of SVCT2 in the NAc. Naloxone (0.4 mM) and bicuculine (150 ng) could block the effect of morphine on the SVCT2, indicating an important role of SVTC2 in the AA release.In conclusion, the NAc is a key nuclear involved in the morphine-induced AA release, as well as the behavioral activity induced by morphine. The mediation may be related to the GABAergic system in the NAc, which may be contributed to the morphine-induced release of AA and GABA in the NAc. Moreover, this action of morphine in the NAc is regulated by the GABAA receptor mediated action of DA afferents from the VTA. In addition, the level of SVCT2 was correspondly changed during the procedure of morphine-induced AA release. Accordingly, several mechanisms are involved in the morphine-induced AA release in the NAc, which might be a potential target for treatment of the neurological disorders induced by morphine-like compounds.更多还原