【作者】 陈爱琴；

【导师】 陈晓春；

【作者基本信息】 福建医科大学 ， 神经病学， 2010， 博士

【摘要】 突触可塑性是神经科学近年来进展最快、取得成果最多的研究领域,其主要表现形式长时程增强(long-term potentiation,LTP)和长时程减弱(long-term depression,LTD)已被公认为是学习记忆活动在细胞分子水平的生物学基础。其中海马的LTP研究最多,诱导方式多样,细胞内机制复杂。行为学实验发现应用NMDA受体拮抗药阻断海马区域的NMDA受体后,大鼠的学习行为能力明显下降,同时海马的LTP不能被成功地诱导产生,表明海马的LTP与学习记忆密切相关。杏仁核作为边缘系统的重要组成部分,主要参与情绪情感有关的学习记忆过程。对杏仁核突触可塑性如LTP及其机制的研究将有助于探讨杏仁核的功能。杏仁核与成瘾药物的共同奖赏通路腹侧被盖区-伏隔核有着密切的解剖结构联系,行为学实验表明杏仁核参与药物成瘾的过程。药物成瘾的核心特征是强迫性用药:即成瘾者失去了对药物摄取和寻觅的控制。在药物成瘾和戒断期间,与药物心理依赖相关的记忆牢固且持久,特别是在吗啡类药物成瘾者中,对药物引起的特殊记忆很难随时间的延长而减弱,而且这种信息的提取同环境密切相关。药物成瘾被普遍认为是一种病理性的强化型记忆,其中长时程增强(LTP)作为学习记忆过程一种主要的细胞内机制,其在成瘾中的作用倍受关注。滥用药物影响药物成瘾相关脑区如腹侧被盖区、伏隔核和海马的LTP,提示这些变化是用药行为产生的结果,而且通过记忆的形式得到加强。杏仁核参与药物成瘾的发生发展过程,然而杏仁核LTP在药物成瘾中的作用目前尚不清楚。我们的实验通过在大鼠离体脑片上记录基底外侧杏仁核(basolateral amygdala, BLA)的场电位,比较不同参数的强直刺激在诱导BLA LTP中的作用,并采用受体阻断剂和细胞通路抑制剂探讨BLA LTP的细胞过程。应用条件性位置偏爱建立吗啡成瘾组、成瘾消退组和复吸组动物模型,观察比较不同组之间BLALTP的差异,探讨杏仁核LTP在药物成瘾中的作用及其机制。大鼠断头后制备脑片,在解剖显微镜下把双极钨刺激电极置于外囊(external capsule,EC),记录微电极的尖端置于杏仁核的基底外侧区。记录电极在脑片表面下约250μm,刺激电极与记录部位之间的距离大约为2 mm。刺激信号由RM6240程控刺激器产生,实验常规刺激的波宽0.1 ms、频率0.1 Hz,场电位通过SWF-2W微电极放大器放大后,应用RM6240系统进行信号的记录、分析和处理。实验中应用不同的参数诱导BLA的LTP,研究不同的刺激方式与LTP诱导之间的关系。通过在灌流液中分别加入NMDA受体阻断剂、PKC和酪氨酸蛋白激酶抑制剂,观察所诱导的LTP的变化,探讨BLA LTP可能的分子机制。实验中观察到在脑片保持良好活性及记录系统噪音幅度小于0.01 mV的前提下,应用国产的微电极放大器及记录系统即可记录到稳定可靠的杏仁核场电位,幅度约为海马CA1场电位的十分之一;在灌流液中加入AMPA受体拮抗剂CNQX 10μM和NMDA受体阻断剂APV 100μM后,场电位几乎完全被阻断,表明杏仁核的突触后电位主要由兴奋性的谷氨酸受体介导。应用间隔10 min的两串高频刺激(100 Hz,每串1s )刺激外囊,在基底外侧杏仁核(BLA)可诱导明显的LTP,两串高频刺激后30min增强的场电位的斜率仍然维持在基础值的146.1±6.9%(n=9,p<0.01)。100Hz的高频刺激是诱导LTP的常用模式,另一种模式θ频率波刺激模拟学习过程锥体细胞典型的放电模式,应用θ频率波刺激更接近生理刺激。每串θ频率波刺激为20个(频率5Hz)短时间高频串脉冲(5个脉冲,频率为100Hz)。同样应用两串的θ频率波刺激诱导BLA LTP,并观察不同的串间隔诱导的BLA LTP的差异。间隔10s的两串θ频率波未能在BLA诱导出LTP;增大串刺激间隔为10min或30min,均可观察到记录的场电位明显增大,增强的场电位持续时间超过30min,串间隔为10min的参数诱导的LTP最明显;但串间隔10min和30min的θ频率波刺激所诱导LTP统计学上没有显著性差异,结果提示神经细胞两个事件的间隔变化可影响突触传递的效率及突触可塑性,从而影响学习记忆的过程,而且可能存在一段最适宜的间隔,间隔太短或太长都将降低学习记忆的效率。EC-BLA的LTP表现为通路特异性,且可被NMDA受体阻断剂APV所阻断。在灌流液中加入蛋白激酶C(PKC)抑制剂chelerythrine chloride,对BLA的基础场电位和配对脉冲比率(paired-pulse ratio, PPR)不产生影响;但在chelerythrine chloride存在的情况下,两串间隔10min的θ频率波刺激未能诱导出LTP;于两串高频刺激10 min之后加入chelerythrine chloride则不能阻断BLA的LTP,结果表明PKC参与BLA LTP的诱导和早期维持。灌流的人工脑脊液中加入酪氨酸蛋白激酶抑制剂genistein也可抑制BLA LTP的诱导,酪氨酸蛋白激酶参与BLA LTP的过程。应用条件性位置偏爱(CPP)建立吗啡成瘾组动物模型,模型建立后的第二天至第四天(成瘾消退之前)制备脑片,应用两串(间隔10s)的高频刺激诱导BLA LTP,比较吗啡成瘾组与对照组LTP的差异。对照组高频刺激后,场电位出现一过性增大,高频刺激30 min后,场电位基本恢复正常,为基础场电位的114.0±6.3% (n=9, P>0.05)。成瘾组高频刺激后,场电位明显增大,并且这种增强作用持续30 min以上,高频刺激后30 min,增强的场电位的斜率仍然维持在基础场电位的154.8±5.3% (n=14, P<0.01)。为观察PKA是否参与成瘾组基底外侧杏仁核LTP的过程,实验中在灌流液中加入PKA抑制剂PKI-(6-22)-amide Tocris(1μmol/L, n=6),其对基础的场电位没有影响,PKI-(6-22)-amide Tocris作用15 min后场电位仍为基础值的99.4±3.8% (n=6, P>0.05)。然而在PKA抑制剂存在的情况下,两串HFS刺激外囊,基底外侧杏仁核诱导的LTP明显减弱。高频刺激后30 min,场电位的斜率为122.2±4.6% (n=6, P<0.01 vs成瘾组)。应用CPP建立吗啡消退组动物模型,与高频刺激前基础值相比,消退组两串HFS仍可诱导出LTP,高频刺激30 min后为基础场电位的128.0±9.3% (n=8, P<0.01),但与成瘾组相比,消退组杏仁核的LTP减弱(P<0.01)。应用半剂量吗啡点燃和强迫冰冷水游泳5 min应激点燃两种方式建立CPP吗啡重现组动物模型。实验中观察到重现组两串HFS又可以在BLA诱导出明显的LTP,高频刺激后30 min,场电位分别为基础场电位的142.7±8.0% (半剂量吗啡重现组,n=7, P<0.01)和143.3±6.0% (强迫冰冷水游泳应激重现组5 min,n=5, P<0.01),但两种点燃方式间没有差异(P>0.05)。实验结果显示串间隔为10min的θ频率波或100 Hz的刺激是诱导BLA LTP的较佳参数。BLA LTP为通路特异性和NMDA受体依赖性,细胞内蛋白激酶C和酪氨酸蛋白激酶参与了BLA LTP的诱导和维持。在吗啡成瘾和发展的过程中,杏仁核LTP的变化不同,成瘾组增强,消退组减弱但与对照组相比仍可诱导出LTP,复吸组又增强,提示成瘾药物所致的BLA LTP并不是持久的、简单的单向变化,而是呈双向的变化趋势,基底外侧杏仁核在药物成瘾过程中既有神经元的适应性变化又有突触可塑性变化。杏仁核参与学习记忆的过程,对杏仁核LTP的特点、性质、表现形式和细胞学机制的研究,有助于了解杏仁核的功能及其在学习记忆中的作用。研究杏仁核LTP与学习记忆在药物成瘾形成和复吸中的作用及其可能的机制,可为评价和干预成瘾的发生发展及复吸提供科学依据。另一方面,药物成瘾可为研究神经系统的突触可塑性机制和作用及大脑的学习记忆过程提供一种模型,将有助于阐明学习记忆的脑内机制。更多还原

【Abstract】 Abstract In recent years, a large body of experimental results on synaptic plasticity has been accumulated in neuroscience. Long-term potentiation (LTP) and Long-term depression are the main forms of synaptic plasticity, which are generally regarded as potential cellular mechanisms underlying learning and memory. LTP in hippocampus is most studied and complicated in cellular mechanism, which is induced by various ways such as drugs or electric stimulus. In behavior experiments the learning capacity decreased significantly and LTP in hippocampus was failed to be induced when the NMDA receptor function was blocked by the application of NMDA receptor antagonist in hippocampus, indicating LTP in hippocampus is closely linked with learning and memory. As an important part of limbic system, Amygdala is mainly involved in emotion-related learning and memory. The research of synaptic plasticity such as LTP in amygdala will help to explore the functions of the amygdala.The amygdala is closely connected with the common reward pathway of ventral tegmental area - nucleus accumbens in drug addiction. Furthermore, behavioral experiments showed that the amygdala was involved in the process of drug addiction. The core character of drug addiction is compulsive drug-abuse: the loss of control over drug-taking and drug seeking. During the period of drug addiction and withdrawal, the memory about drug-related psychological dependence is solid and lasting. Especially in the morphine-type drugs addicts, the special memories about drug-related information is hard to weaken with time and the extraction of the information is closely related with the environment. Drug addiction is widely considered to be an enhanced memory and the role of LTP in drug addiction is well-focused as a cellular mechanism of learning and memory. Drug abuse affected LTP in drug addiction-related brain areas such as the ventral tegmental area, nucleus accumbens and hippocampus, suggesting that these changes were caused by the abuse of drug and enhanced by the form of memory. Amygdala is involved in the occurrence and development of drug addiction, however, the role of amygdala LTP in drug addiction is unclear.In our experiments we compared the effects of different patterns of titanic stimulus on the induction of LTP in basolateral amygdala (BLA) by recording field potentials in BLA in rat brain slices in vitro. We explored the cellular process of BLA LTP by bath application of receptor antagonists and cell pathway inhibitors. Then we established the animal models of morphine addiction, extinction and reinstatement groups by using conditioned place preference. Studies were done to compare the differences of BLA LTP in brain slices from different groups to explore the role and mechanism of BLA LTP in drug addiction.Brain slices were prepared after the decapitation of rats. The sharpened tungsten bipolar stimulating electrodes and recording microelectrode tips were visually positioned in external capsule (EC) and in the basolateral region of the amygdala individually using a dissecting microscope. The tips of field potential electrodes were located about 250μm under the surface of the slices and the distance of stimulating electrode and recording electrode was about 2 mm. Stimuli were delivered using a RM6240 programmable stimulator. Single 0.1-ms monophasic square pulses were applied continuously throughout the experiment at 0.1 Hz. The field potentials were amplified with an SWF-2W amplifier. On- and off-line data acquisition and analysis was carried out using RM6240. Different parameters were applied to induce LTP in BLA to understand the relationship between stimulating pattern and the induction of LTP. To explore the possible molecular mechanism of BLA LTP, N-methyl-D-Aspartate (NMDA) receptor antagonist, the inhibitors of protein kinase C (PKC) and tyrosine protein kinase were added to the perfusion and then LTP was induced. In our experiments in the prerequisite of slices in good activity and recording system noise level under 0.01mv, field potentials in BLA were evoked stably with stimulating electrodes placed in the external capsule (EC) using domestic-made microelectrode amplifier and recording system. The amplitudes of field potentials in BLA were about one-tenth of that in hippocampus CA1. The fielding potentials in BLA were almost totally blocked in the presence of AMPA antagonist CNQX (10μM) and NMDA receptor antagonist APV (100μM), indicating excitatory post synaptic potentials in BLA are glutamate receptor-dependent. LTP in BLA was induced by application of two high frequency stimulations (HFS, 100 Hz, 1s each train) of internal 10 min to external capsule. The theta burst stimulation protocol was applied because it mimics the typical firing mode of pyramidal cells during learning.The slope of field potentials 30 min after HFS was 146.1±6.9%(n=9,p<0.01)) of the initial baseline values. High-frequency stimulation of 100Hz is the common pattern to induce LTP. Theta burst stimulation (TBS) is another model being often used to induce LTP because it mimics the typical firing mode of pyramidal cells during learning and is closer to physiological stimulus. The parameter of TBS is a brief, high-frequency pulse train (5 pulses at 100 Hz) given at the theta-rhythm (5Hz) for 4 sec. Experiments were done to compare the effects of different intervals of two TBS on the expression of LTP in BLA. Two TBS of 10 s interval failed to induce LTP in BLA. The interval of two TBS increased to 10 min and 30 min, individually, both types of stimulations enhanced f-EPSPs in BLA and the enhanced f-EPSPs lasted more than 30 min. Two TBS of 10 min interval is better in the induction of LTP in BLA, however, there is no significant difference in statistics between the interval of 10 min and 30 min. LTP in BLA was input specific and was blocked by N-methyl-D-Aspartate (NMDA) receptor antagonist APV. The effect of protein kinase C (PKC) on LTP was then determined using PKC inhibitor chelerythrine chloride.Bath application of chelerythringe chloride had no effect on basic field potentials and paired-pulse ratio (PPR). However, in the presence of chelerythrine chloride, two TBS failed to induce LTP. In contrast, bath application of chelerythrine chloride 10 min after the second TBS didn’t affect the maintenance of LTP in BLA. Furthermore, LTP in BLA induced by two TBS of 10 min and 30 min interval was also blocked by bath application of tyrosine protein kinase (TPK) inhibitor genistein.We established the animal models of morphine addiction by using conditioned place preference. Brain slices were prepared two to four days after the establishment of morphine addiction (before the extinction) and BLA LTP was induced by using two 10 s-interval HFS of 100 Hz in slices. Then we compared the differences of BLA LTP between addiction group and control. In control, application of two HFS only induces a short-term synaptic potentiation (STP). The field potential almost returned to normal and the slope was 114.0±6.3% (n=9, P>0.05) of the initial baseline values 30 min after HFS.In addiction group, the field potential increased significantly after two HFS and the enhanced field potential lasted more than 30 min. The slope of field potential was 154.8±5.3% (n=14, P<0.01) of the baseline 30 min after HFS. To observe the role of protein kinase A (PKA) in BLA LTP in addiction group, the PKA inhibitor PKI-(6-22)-amide Tocris(1μmol/L) was added to the perfusion. The PKA inhibitor had no effects on basal synaptic potential and the field potential was still 99.4±3.8% (n=6 P>0.05) of the basal values after 15 min perfusion. However, in the presence of PKI-(6-22)-amide Tocris, the BLA LTP induced by two HFS attenuated obviously and the slope of field potential was 122.2±4.6% (n=6) of baseline.The extinction model of morphine addiction was established by application of CPP. Two 10 s-interval HFS induced LTP in slices from extinction model and the slope of field potential was 128.0±9.3% (n=8 P<0.01) of baseline 30 min after HFS. However, BLA LTP attenuated in extinction group compared to the addiction group. To establish the relapse model, we adapted two ways of semi-dose morphine and forcing-swimming for 5 min in icy-cold water to reinstate extinguished CPP. Two HFS again obviously induced BLA LTP in slices from the relapse model and there was no significant difference between two ways of reinstatement. The field potential was 142.7±8.0% (n=7, P<0.01) of baseline in semi-dose morphine reinstatement group and 143.3±6.0% (n=5 P<0.01) in forcing-swimming reinstatement group individually.The results showed that the 10 min-interval stimulation of two TBS or 100 Hz was the better pattern of inducing BLA LTP in slices. BLA LTP is input-specific and NMDA receptor-dependent. Furthermore, intracellular protein kinase C and tyrosine protein kinase were involved in the induction and maintenance of BLA LTP. LTP in amygdala changed with the occurrence and development of morphine addiction. BLA LTP enhanced in morphine addiction model but attenuated in extinction group (which was potentiated compared to control) and strengthened again in relapse model, suggesting that the change of BLA LTP caused by addictive drugs is not durable, simple one-way but two-way trend. The changes in basolateral amygdala in the process of drug addiction include the adaptive changes of neurons and synaptic plasticity. Amygdala is involved in the process of learning and memory. The study of LTP in amygdala in the character, nature, pattern of manifestation and cellular mechanism will help to understand the function of amygdala and its role in learning and memory. The research of the role and mechanism of LTP of amygdala and learning and memory in drug addiction and relapse will prodive a scientific basis for the evaluation and intervention of addiction development and relapse. On the other hand, drug addiction may be a model to study synaptic plasticity in nervous system and the process of learning and memory, helping to illuminating the brain mechanism of learning and memory.更多还原