【作者】 谢桂琴；

【导师】 袁孝如；

【作者基本信息】 南京医科大学 ， 药理学， 2009， 博士

【摘要】 药物滥用是现今世界范围内危害人类健康、危及社会安定的重要医学和社会学问题,成瘾物质的奖赏和增强作用使患者不顾风险地吸食,尤其是欣快和解忧的快感在中枢留下清晰记忆导致患者复吸。复吸通常发生在当成瘾者重新遇到和他以前服药时相关的人、事、地点等暗示信号的时候,这提示成瘾过程中可能出现了由药物诱导的行为和神经元突触可塑性的病态稳定形式,这种突触可塑性可视为学习记忆的一种特殊形式:在药物作用下,神经元和神经网络发生可塑性变化从而产生特定的习得性行为,并形成记忆痕迹,储存在脑的奖赏和记忆系统里。因此,成瘾也被认为是一种病态的学习和记忆过程,以失控的主动寻药和强迫性用药为特征的脑和行为方式的紊乱。酒精是迄今使用时间最长、范围最广泛的成瘾药物,但其中枢作用机制仍留有许多未知有待阐释。中枢神经系统对成瘾物质大多产生两种反应:神经适应性反应和突触可塑性变化。然而,过去几十年的研究大多集中在中枢对成瘾物质神经适应性反应方面,而较少关注突触可塑性变化以及联合型学习机制。长时程增强(long term potatiation,LTP)被公认为突触传递易化重要的电生理指标,作为持久形式的突触可塑性反映突触水平上信息储存过程,是记忆巩固过程中神经元生理活动的客观指标之一。虽然成瘾时神经元突触长时改变与学习记忆系统的LTP很相似,但前者诱导的LTP是病理性的。围绕在大脑皮层、基底神经节、丘脑间,并与多种神经结构形成复杂神经网络的纹状体近年来被认为是药物滥用的主要靶区,有研究认为,从中脑VTA向背侧纹状体投射的多巴胺系统涉及到学习记忆形成和行为习惯的执行。因此,对背侧纹状体的观念发生了重大改变,认为它也是成瘾习惯形成的中心脑区:以其中储藏的多巴胺作为信号固化模式可导致患者向强迫性、习惯性行为模式转化,完成从偶而用药向强迫性服药行为转化。近年被分析出来的一个信号级联MAPK级联(MAPKkk、MAPKk、MAPK)广泛分布于细胞浆内,具有丝氨酸和苏氨酸双重磷酸化能力,由不同的细胞外刺激介导细胞表面至细胞核激活,是细胞内重要的信号传递者,现有较多的研究只是注意到它与细胞生长、分化、增殖和肿瘤形成有关,在海马上的研究发现MAPK能参与调控神经元多种活动包括学习记忆。但MAPK信号级联是否在药物成瘾的长时程记忆中起重要作用则有待进一步研究。因此,本研究的目的在于阐释酒精如何影响背侧纹状体的突触可塑性:首先观察背侧纹状体突触可塑性的表现及其与膜受体的关系;其次研究酒精是否对其产生影响以及与酒精剂量的关系;最后阐明酒精影响背侧纹状体突触可塑性的细胞内机制是否涉及MAPK信号通路。第一部分背侧纹状体长时程增强(LTP)的诱导及其与NMDA受体的关系目的:研究强直刺激(HFS)在背侧纹状体所诱导的突触可塑性表现及其与膜NMDA受体的相关性。方法:SD大鼠制备脑片47张(大鼠15只),随机分为4组:对照组(正常ACSF灌流)脑片25张;无Mg2+组(去Mg2+ACSF灌流)脑片8张;MK801组(MK801 20μM处理脑片)脑片9张;AP5组(AP5 50μM处理脑片)脑片5张。脑片均施加HFS,参数为:4串,串长1s,频率100Hz,每串间隔20s,刺激波宽0.1ms,刺激强度为最大刺激强度的1.2-1.4倍,约为180-250μA,观察各组可塑性表现。结果:各组脑片HFS之后第60 min的记录到的PS幅值增加,在对照组为刺激前的145±14 %,25张脑片中15张表现为LTP,10张无改变;在无Mg2+组为刺激前的126±5 %,8张脑片中6张表现为LTP,2张无改变;在MK801组为刺激前的104±5%,均未表现出LTP;在AP5组幅值为刺激前的105±4%,未表现出LTP。结论:HFS可在背侧纹状体稳定地诱导出LTP,这种LTP经由膜受体NMDA受体介导。第二部分不同浓度酒精对背侧纹状体PS和LTP的影响目的:观察酒精对背侧纹状体PS以及LTP的影响及其与酒精浓度的关系。方法:SD大鼠制备脑片77张(大鼠36只),随机分为5组:对照组(正常ACSF灌流脑片)脑片25张;22 mM组(以22 mM酒精灌流脑片)5张予测试刺激,6张予强直刺激;44 mM组(以44 mM酒精灌流脑片)5张予测试刺激,7张予强直刺激;66 mM组(以66 mM酒精灌流脑片)7张予测试刺激,8张予强直刺激;88 mM组(以88 mM酒精灌流脑片)9张予测试刺激,5张予强直刺激。HFS的参数同实验一,观察各组PS以及LTP的变化。结果:脑片用不同浓度酒精灌流和洗脱处理,施以测试刺激,第60 min的PS幅值在22、44、66、88 mM组分别为灌流前的100.2±1.3%、97.6±3.2%、95.4±3.0%、91.2±6.3% ;脑片以不同浓度酒精灌流处理15 min,施以HFS之后第60 min的PS幅值在22、44、66、88 mM组分别增加为刺激前的124±10%、113±13%、98±5%、94±7%。结论:酒精在背侧纹状体对HFS所致LTP的抑制呈剂量相关性,酒精本身对PS的影响与这种抑制无关,但可能影响了LTP的大小。第三部分酒精抑制背侧纹状体LTP与MAPK信号通路关系的研究目的:研究酒精对背侧纹状体LTP的抑制与细胞内MAPK(ERK和JNK)信号通路的关系。方法:SD大鼠制备脑片162张(大鼠72只),随机分9组:对照组(正常ACSF灌流脑片)脑片46张;DMSO溶剂对照组脑片14张;U0126灌流组脑片18张;22 mM酒精灌流组脑片8张;44 mM酒精灌流组脑片8张;66 mM酒精灌流组脑片40张;88 mM酒精灌流组脑片8张;MK801灌流组脑片12张;SP600125灌流组脑片8张,采用电生理技术记录并在电生理实验后收集脑片以western blotting方法检测蛋白含量,HFS的参数同实验一。结果:给予强直刺激后的第10分钟,与刺激前相比,对照组脑片ERK磷酸化明显增强,并在第60分钟恢复为正常水平;而用ERK抑制剂U0126处理脑片,在抑制ERK活性同时也阻止了强直刺激后LTP的诱导。如果不施加强直刺激,不同浓度酒精灌流脑片并不会引起ERK基础磷酸化水平的改变;而在强直刺激后10 min,22、44 mM的酒精处理组ERK磷酸化与对照组相比无明显区别;66、88 mM的酒精灌流可以抑制强直刺激所致之ERK磷酸化;并且在强直刺激后的第60 min,66 mM酒精处理组ERK磷酸化恢复为正常水平。以MK801处理脑片可以抑制强直刺激后第10min的ERK磷酸化,这种效果在第60 min消失,ERK磷酸化恢复为正常水平。另一方面,在背内侧纹状体,以JNK抑制剂SP600125灌流脑片30 min(30 min pre-HFS),并不影响LTP的诱导。在不同条件下检测JNK活性,发现对照组pJNK(46kD)和pJNK(54kD)的水平在强直刺激前、强直刺激后的10 min、20 min、60 min都无明显改变;在66 mM酒精处理的脑片中,强直刺激前后其JNK活性与对照组相比也无明显区别。结论:MAPK信号通路家族中ERK激酶参与了经NMDA受体介导之酒精对纹状体HFS-LTP诱导的剂量相关性抑制效应,而JNK激酶则与此无明显相关性。综上所述,本文工作的主要创新之处在于:1、本研究发现,与习惯形成密切相关的背侧纹状体,是酒精作用的中枢靶区之一;HFS可在纹状体稳定地诱导LTP,这一LTP是经膜NMDA受体介导的。2、观察了不同浓度的酒精(22--88 mM)呈剂量依赖性抑制LTP的诱导,其中66 mM抑制效果最强和稳定。3、用western blot方法观察了LTP诱导和维持的不同时期表达的变化,以及酒精对其的影响,发现酒精是通过降低HFS所致MAPK/ERK活性增强进而抑制LTP的诱导,而与MAPK/JNK活性无关。更多还原

【Abstract】 Ethanol is a widely used and abused drug that affects learning and behavior and can induce an acute amnestic state. Compared to the effects of other addictive substances such as cocaine and nicotine, the effects of ethanol are more diffuse, including diverse documented effects on various ligand-gated and voltage-gated channels. Consequently, the mechanisms underlying ethanol’s actions on the central nervous system are difficult to pin down, and a greater understanding of these mechanisms might provide a foundation for new treatments for alcoholism.Clinical and laboratory observations have proposed that addiction represents the pathological usurpation of neural processes that normally serve reward-related learning. Long-term potentiation (LTP) enhances learning and long-term memory by strengthening the synapse, the point through which nervous impulses pass from one neuron to another. Long-term depression (LTD) is essentially the opposite, weakening the neuronal synapse. Therefore, LTP and LTD have become an important focus of addiction research. Reward-guided behaviors are often attributed to the cortico-basal ganglia networks coursing through the neostriatum. The dorsal striatum has been proposed to contribute to the formation of drug-seeking behaviors, leading to excessive and compulsive drug usage, such as addiction. However, the involvement of the dorsomedial striatum (DMS) in acute drug abuse has not received much attention, and the mechanisms underlying the effects of acute ethanol on corticostriatal plasticity still remain unclear.Extracellular signal regulated protein kinases (ERKs), which are mitogen activated protein kinases (MAPKs) that are 44 and 42 kD, are involved in diverse forms of neuronal plasticity. Recently, evidence has pointed to ERKs as having a role in the LTPs that occur in the CA1 region of the hippocampus. The MAPK?ERK signal transduction pathway follows the binding of growth factors to receptors on the cell’s surface to its intracellular responses. Several studies suggested the ERK signal transduction pathway as a potential cellular target of ethanol. Alterations in ERKs could play an important role in the chronic alcoholinduced changes in synaptic plasticity. These findings raise the possibility that ERK activation may contribute to the striatal LTP induction and to the effects of acute ethanol on corticostriatal plasticity. To test this possibility, we studied the high-frequency afferent stimulation (HFS) - induced ERK activation in acute ethanol-treated slices from the rat DMS. We also examined the effects of the N-methyld- aspartic acid receptor (NMDAR) antagonist MK801 on ERK activation in this brain region.c-Jun N-terminal kinase (JNKs), another kind of MAPKs, are involved in diverse forms of neuronal plasticity, too. We tested the alteration of JNK activation in this work, too.Part I Long-term potentiation induced by HFS in striatum of ratAIM:To investigate striatal synaptic plasticity involved in instrumental learning.METHODS:The stimulation evoked population spikes (PS) were recorded from the dorsomedial striatum (DMS) slices of rat using the extracellular recording technique. The LTP in DMS slices was induced by high-frequency stimulation (HFS).RESULTS:HFS evoked obvious LTPs both in the slices incubated with ACSF and ACSF without Mg2+. MK801 and APV, N-methyl-d-aspartic acid receptor (NMDAR) antagonists, inhibited the induction of striatal LTPs.CONCLUSION:The LTP of the PS in the DMS is, at least partly, mediated by NMDARs.Part II Effects of different concentrations of ethanol on the striatalLTPAIM: The objective of this investigation was to study the effects of acute perfusion of ethanol on PS and long term potentiation (LTP) to elucidate the mechanisms of addictive drugs in the striatum.METHODS: The stimulation evoked population spikes (PS) were recorded from the dorsomedial striatum (DMS) slices of rat using the extracellular recording technique. The LTP in DMS slices was induced by high-frequency stimulation (HFS).RESULTS: Clinically relevant concentrations of ethanol (22 to 88 mM) dose-dependently attenuated the HFS-induced striatal LTP in this brain region. 22, 44, 66 and 88 mM of ethanol induced a change in PS amplitude by 0.2±1.3% (p = 0.37, n = 5), -2.4±3.2% (p = 0.07, n = 5), -4.6±3.0% (p < 0.05, n = 7) and -8.4±6.3% (p < 0.05, n = 9), respectively. All these changes returned to the baseline levels after washout of ethanol. The PS amplitude before HFS (baseline PS) was compared to that recorded 60 min after HFS. The PS amplitudes at 60 min after HFS increased to 145±14% of the baseline value (n = 14) in the slices under control conditions. After the perfusion of 22, 44, 66 and 88 mM ethanol, the PS amplitudes at 60 min post-HFS were 124±10% (n = 6), 113±13% (n = 7), 98±5% ( n = 8), 94±7% (n = 4) of the pre-HFS baseline values, respectively.CONCLUSION: Ethanol attenuated the HFS-induced LTP in a dosedependent manner in this brain region.Part III Effects of ethanol on the striatal LTP involved in the MAPKsactivationAIM: To study the effects of acute perfusion of ethanol on long term potentiation (LTP) as well as to elucidate the mechanisms of addictive drugs in the striatum. In addition, we investigated the contribution of both ERKs and JNKs signaling pathways to corticostriatal LTP induction.METHODS: The stimulation evoked population spikes (PS) were recorded from the dorsomedial striatum (DMS) slices of rat using the extracellular recording technique. The LTP in DMS slices was induced by high-frequency stimulation (HFS). Both the ERKs and JNKs levels of the DMS were assessed with the Western blot technique.RESULTS: U0126, the inhibitor of ERK, eliminated or significantly attenuated the LTP induced by HFS of the PS in the DMS. MK801 and APV, N-methyl-d-aspartic acid receptor (NMDAR) antagonists, inhibited the induction of striatal LTP, and HFS-induced ERK activation decreased in the slices treated with MK801 in the DMS. Clinically relevant concentrations of ethanol (22 to 88 mM) dose-dependently attenuated the HFS-induced striatal LTP and ERK activation in this brain region. On the other hand, SP600125, the inhibitor of JNK, had no effect on the striatal LTP. Furthermore, ethanol (66 mM) did not show any effect on JNK activation before or after HFS in the DMS.CONCLUSION: The LTP of the PS in the DMS is, at least partly, mediated by the ERK (but not JNK) pathway coupling to NMDARs. Ethanol attenuated the HFS-induced, ERK-mediated LTP in a dose-dependent manner in this brain region.In summary, the present study reports for the first time that ethanol may change the synaptic plasticity of corticostriatal circuits underlying the learning of goal-directed instrumental actions, which is mediated by an intracellular ERK signaling pathway associated with NMDARs.更多还原