吗啡给药及戒断期间大鼠眶额叶脑区功能活动特征户外环境下自由活动猕猴顶叶及海马神经元单位放电的胞外记录方法
文献类型：学位论文
| 作者 | 孙宁磊 |
| 学位类别 | 博士 |
| 答辩日期 | 2007-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 马原野 |
| 关键词 | 药物滥用 眶额叶 锰离子增强的核磁共振成像 灌流 心理渴求 脑电 |
| 其他题名 | Dynamic changes in orbitofrontal neuronal activity in rats during opiate administration and withdrawal |
| 学位专业 | 动物学 |
| 中文摘要 | 已有的研究表明，眶额叶在解剖上与现在已知的药物滥用相关的脑区是紧密联系在一起的。例如，眶额叶在药物滥用和强迫性重复行为中起作用，且随着脑成像技术的应用，越来越多的证据表明眶额叶参与了药物滥用。但是我们并不了解在阿片给药和戒断期间眶额叶脑区活动是如何变化的。因此，我们在实验中采用了Mn2+增强的核磁共振成像（Manganese-enhanced magnetic resonance imaging，MEMRI，4.7T）技术和脑电（EEG）记录的方法，以研究大鼠眶额叶在给与阿片类药物（盐酸吗啡）以及戒断过程中的动态变化。 MEMRI是一种近年才发展起来的新型技术。研究表明，Mn2+是Ca2+的类似物，可以通过Ca2+通道进入兴奋性的神经元里面并结合到胞内的蛋白质和核酸上的Ca2+和Mg2+结合位点上 (MILDVAN and COHN, 1963; EISINGER et al., 1965)。另外，Mn2+的顺磁性也为它成为核磁共振成像的造影剂提供了前提条件。可是成功应用MEMRI的前提就是要在适当的时间把合适剂量的Mn2+传递到靶点上。因此，Mn2+在注射到靶点后，是否能够在有效的时间内反映大脑活动的变化就成为一个非常重要并且在技术上较为棘手的问题。在给实验大鼠脑区微量注射Mn2+（80mMol/L，200nl）的同时，通过微量注射兴奋性神经递质谷氨酸（Glu 0.5mM/L）或抑制性递质γ-aminobutyric acid（GABA 0.5M/L）以改变靶点神经元兴奋性的方法，检测Mn2+能否反映脑区的活动变化。另外，我们随机选取实验动物，分别在注射Mn2+ 3小时、5小时和8小时后对三组大鼠（n=5）进行10％福尔马林灌流，并且通过观察大鼠眶额叶脑区Mn2+强度的变化来研究最佳的灌流时间。我们的实验结果表明，Mn2++Glu组的右侧脑区/左侧脑区的Mn2+亮度比Mn2+空白对照组增加了20％（p=0.016, student t-test, *p <0.05），也远大于Mn2++GABA组（p=0.047, *p<0.05）。结果表明，当神经元被兴奋的时候，较多的Mn2+可以通过Ca2+通道进入兴奋的神经元内，使得Mn2+的成像亮度增加。由于Mn2+成像亮度的增加可以反映神经元的兴奋活动，因此可以显示出靶点区的脑活动。另外，在研究灌流时间对Mn2+亮度影响的实验中发现，注射Mn2+ 5小时后灌流得到的信噪比分别比注射Mn2+3小时（p＝0.055）和8小时（p=0.004，*p<0.05）高出24％和32％。总之，我们采用微量注射Mn2+（80mM/L,200nl）后5小时用10%福尔马林心脏灌流的方法获得了较好的结果。另外在试验中我们首先观测了大鼠吗啡戒断后的行为学指标和检测大鼠戒断后条件化位置偏好的程度。实验结果表明大鼠可以建立非常明显的条件化位置偏好，但在湿狗抖等行为学指标上无明显症状。这说明大鼠对于吗啡(10mg/kg, 一天两次，持续12天)形成了明显的心理依赖而无明显的生理依赖。此外，MEMRI的结果表明，在吗啡给药的第1天和第6天，大鼠眶额叶的Mn2+强度与空白对照组相比有显著的降低( one-way ANOVA, Post Hoc Dunnett’s C Tests), F (6,28)=7.242, P<0.001)；而在戒断第3天又恢复到正常水平，在戒断第5天和第7天Mn2+强度跟空白对照组相比没有显著性差别(one-way ANOVA, *p<0.05)。脑电（EEG）的结果表明，急性吗啡诱导的gamma波段的EEG显著降低(Two-way ANOVA, F(1,10)=13.626,p=0.006)。然而在戒断第1天gamma波段的EEG与空白对照组相比是增加的。在戒断第3天和戒断第5天，gamma波段的EEG与空白对照组相比也有显著性增强。以上研究结果表明：大鼠眶额叶脑区的动态变化与整个吗啡给药和戒断过程是密切相关的；此外，MEMRI在探讨药物滥用以及成瘾等机制上有很大的应用前景。 |
| 英文摘要 | As we as known, the orbitofrontal cortex (OFC) is anatomically connected with brain areas known to be involved in the reinforcing effects of drugs of abuse. Several sources of information suggest that the orbitofrontal cortex plays a role in behavioral disorders such as drug abuse and compulsive repetitive behavior. Using brain imaging techniques, previous studies have provided imaging evidence of the involvement of the orbitofrontal cortex in drug abuse.However, how the dynamic activity in OFC changes during opiate administration and withdrawal period have not been investigated. We first tested the effects of opiates and drug craving with behavior test and conditioned place preference paradigm, using manganese-enhanced magnetic resonance imaging (MEMRI) and traditional EEG recording technique in rats. MEMRI is one new develping technique. In principle, the approach relies upon the fact that Mn2+ can enter excitable cells using some of the same transport systems as Ca2+ through voltage-gated divalent cation channels, which are ubiquitous in neurons and it can bind to a number of intracellular sites because it has high affinity for Ca2+ and Mg2+ binding sites on proteins and nucleic acids(MILDVAN and COHN, 1963; EISINGER et al., 1965). Furthermore, paramagnetic forms of manganese ions are potent MRI relaxation agents. So it is necessary to put Mn2+ into region of interst (ROI) in proper dose and at effective time. However, the question is how to keep the brain activity after Mn2+ injection until effective time. We first injected Mn2+ （80mMol/L，200nl）and transmitters, glutamine（0.5mM/L） and GABA（0.5M/L）, into ROI to alter brain activity , to test whether Mn2+ could indicate the brain activity or not. Furthermore, rats were chosen by random to be perfused 3 hour, 5 hour and 8 hour after Mn2+ injection into interesting area, in order to obtain the best ratio of signal/noise of Mn2+ changes. Our data shows that Mn2+ intensity ratio of right/left side in Mn2++Glu group is 20% higher than in Mn2+control group（p=0.016, student t-test, *p <0.05）and also significantly higher than in Mn2++GABA group （p=0.047, *p<0.05）. The results indicate that Mn2+ could enter the more excitable neurons, which elevates the level of Mn2+ intensity in interesting area and in further proves the fact that MEMRI can the exciting brain activity in interesting area. In addition, it is clearly showed that Mn2+ intensity in the ratio of signal/noise 5 hours after perfusion is 24% higher (p＝0.055）and 32% (p=0.004，*p<0.05） than 3 hr perfusion and 8 hr perfusion individually. In conclusion, we injected Mn2+（80mM/L,200nl）into interesting area and perfused rats by 10% formalin 5 hours after perfusion and obtained good results. In our experiements, we obtained the behavior index and tested the CPP during morphine withdrawal. Our data indicated that rats had significant place preference but had not significant withdrawal synodrome, such as wet-dog shake and twisting. Those results showed rats exposured in morphine (10mg/Kg, twice one day) had significant psychological dependence on morphine ,not physical dependence. T1-weighted 2D MRI (4.7T) was used after unilateral injection of MnCl2 (200nL, 80mM) into the right orbitofrontal cortex. The MEMRI data suggested that the Mn2+ intensity in OFC significantly decreased compared with the saline control group on the 1st and 6th day of morphine administration (one-way ANOVA, F=7.242, n=5; *p<0.05, **p<0.01 separate days 1 and 6 and n=5). During withdrawal, the Mn2+ intensity in OFC decreased significantly on the 1st day after withdrawal and recovered normal level on the 3rdday after withdrawal. In addition, the Mn2+ intensity on the 5th and 7th day after morphine withdrawal was not significantly different compared with the control group (one-way ANOVA,*p<0.05). Our EEG data showed that acute morphine could induce significant decreases of gamma-band EEG activity, compared with saline injection during all administration period(Two-way ANOVA, F(1,10)=13.626,p=0.006) . However, the OFC gamma-band EEG power began to rise on the 1st day after withdrawal. The increase in gamma-band EEG power achieved significance compared to the control group on the 3rd and 5th day after withdrawal. In conclusion, our study shows that dynamic changes in brain activity are amenable to study across the addiction and withdrawal process, and we also believe that the MEMRI paradigm has particular promise for exploring brain function underlying behavioral disorders such as addiction. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6269]  |
| 专题 | 昆明动物研究所_认知障碍病理学 |
| 推荐引用方式 GB/T 7714 | 孙宁磊. 吗啡给药及戒断期间大鼠眶额叶脑区功能活动特征户外环境下自由活动猕猴顶叶及海马神经元单位放电的胞外记录方法[D]. 北京. 中国科学院研究生院. 2007. |
入库方式： OAI收割
来源：昆明动物研究所
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