大鼠苍白球HCN通道的电生理学和行为学研究

发布时间：2018-05-28 18:20

本文选题：苍白球 + HCN通道　；参考：《青岛大学》2012年硕士论文

【摘要】：苍白球作为基底神经节间接环路的重要中继核团,在正常和病理状态下对机体运动功能起重要的调节作用。研究表明超级化激活环核苷酸门控(hyperpolarization-activated cyclic nucleotide-gated, HCN)通道在苍白球自发起搏电活动中发挥重要作用。形态学研究证实苍白球有HCN通道表达。离体脑片膜片钳研究揭示HCN通道参与调节苍白球神经元自发放电活动。目的：探讨HCN通道阻断剂ZD7288对正常大鼠和6-羟基多巴胺(6-hydroxydopamine,6-OHDA)帕金森病模型大鼠苍白球神经元的在体电生理效应,ZD7288对氟哌啶醇僵直大鼠整体姿势行为的影响,以及正常和帕金森病模型大鼠苍白球神经元HCN通道各亚型的表达情况。方法：本实验采用多管微电极在体细胞外电生理记录、帕金森病大鼠模型的制备、埋管术以及免疫组织化学染色等实验方法。结果：1.在正常大鼠苍白球记录到的35个神经元中,有16个(45.7%)神经元在微量压力注射0.5mM ZD7288后自发放电频率由12.0±2.1Hz降低至6.5±1.3Hz,平均降低53.9±6.7%(P0.001)；另外16个(45.7%)神经元可以被0.5mM ZD7288兴奋,自发放电频率由8.9±0.9 Hz升高到16.6±1.9Hz,平均升高90.3±14.8%(P0.001)。2.在6-OHDA帕金森病模型大鼠损毁侧记录到的27个苍白球神经元中,微量压力注射0.5mM ZD7288可以使其中10个(37.0%)神经元的自发放电频率由7.8±2.4Hz降低到3.1±1.3Hz,平均降低70.2±7.5%(P0.01)；另外15个(55.6%)苍白球神经元在微量压力注射0.5mMZD7288后自发放电频率由7.8±1.4 Hz升高至17.1±3.0Hz,平均升高149.8±29.8%(P0.001)。3.在6-OHDA帕金森病模型大鼠损毁对侧记录到的28个苍白球神经元中,微量压力注射0.5mM ZD7288可以使其中12个(42.9%)神经元的自发放电频率由12.8±2.4Hz降低到7.1±1.4Hz,平均降低46.1±4.6%(P0.01)；另外10个(35.7%)苍白球神经元在微量压力注射0.5mM ZD7288后自发放电频率由7.9±2.4Hz升高至14.3±3.0Hz,平均升高130.7±34.2%(P0.01)。在6-OHDA帕金森病模型大鼠,ZD7288对损毁侧苍白球神经元的抑制效应较损毁对侧明显增强,差别有统计学意义(P0.05)。4.应用D-AP5(0.8mM)和CNQX(1.0mmM)的混合物观察谷氨酸能突触传递是否参与ZD7288介导的兴奋效应。在17个苍白球神经元中,单独给予D-AP5和CNQX的混合物不引起苍白球神经元兴奋性的改变。在D-AP5和CNQX存在的情况下,ZD7288仅兴奋了3个神经元(17.7%),兴奋细胞率较不给予D-AP5和CNQX时(45.7%)明显降低(P0.05)。在D-AP5和CNQX存在的情况下,有8个苍白球神经元兴奋性未发生明显改变,无反应细胞率(47.0%)较不给予D-AP5和CNQX时(8.6%)明显升高(P0.01)。5.在行为学实验中,腹腔注射氟哌啶醇能使大鼠产生相对稳定的僵直症状,单侧苍白球微量注射ZD7288可致帕金森病僵直模型大鼠出现不同的偏转行为。其中在实验组9只大鼠中,有4只大鼠表现出明显的对侧偏转行为,与生理盐水组相比有统计学意义(u=132.500,P0.001)；另外5只表现为明显的同侧偏转行为,与生理盐水组相比有统计学意义(u=9.000,P0.001)。6.在免疫组织化学染色实验中,正常及帕金森病模型大鼠苍白球内均观察到HCN1、HCN2和HCN4的表达,其中以HCN2亚型为主,HCN1及HCN4呈少到中等量表达。三种亚型在苍白球神经元胞体及神经纤维上均有表达。HCN1亚型在神经元胞体表达较多；HCN2亚型主要表达于神经纤维,胞体表达较少；而HCN4亚型在神经元胞体及神经纤维上的表达量相当。结论：电生理学研究结果显示ZD7288阻断HCN通道后可改变正常及帕金森病模型大鼠苍白球神经元的兴奋性,分别产生放电频率降低和升高的效应。ZD7288引起的兴奋效应可能与谷氨酸传递有关。行为学实验则进一步揭示苍白球微量注射ZD7288能够诱导氟哌啶醇所致的帕金森病僵直大鼠出现不同的偏转行为,提示可能与ZD7288对苍白球神经元的不同电生理效应有关。形态学研究显示正常及帕金森病模型大鼠苍白球中均有HCN1、HCN2、HCN4受体的表达。本实验结果为深入探讨苍白球HCN通道在帕金森病发病机制中的作用提供了一定的理论和实验依据。
[Abstract]:The globus pallidus, an important relay nucleus of the indirect loop of the basal ganglia, plays an important role in regulating the movement function of the body in normal and pathological conditions. The study shows that the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel is an important role in the self initiated pacing activity of the globus pallidus. The morphological study confirmed that the globus pallidus had HCN channel expression. The study revealed that the HCN channel was involved in regulating the spontaneous discharge activity of the pallid bulb. Objective: To explore the body power of the HCN channel blocker ZD7288 on the globus pallidus neurons in the normal rats and the 6- hydroxy dopamine (6-hydroxydopamine, 6-OHDA) Parkinson's disease model rats. The effects of physiological effects, ZD7288 on the overall posture of haloperidol rats and the expression of the HCN channel subtypes in the white ball neurons of normal and Parkinson's disease rats. Methods: the electrophysiological record of multi tube microelectrodes, the preparation of the rat model of Parkinson's disease, the buried tube and the immunohistochemical staining were used in this experiment. Results: 1. of the 35 neurons recorded in normal rat pallidus, 16 (45.7%) neurons were reduced to 6.5 + 1.3Hz from 12 + 2.1Hz to 6.5 + 1.3Hz after micro pressure injection of 0.5mM, and the average decreased 53.9 + 6.7% (P0.001), and the other 16 (45.7%) neurons could be excited by 0.5mM ZD7288 and were self distributed. The electrical frequency increased from 8.9 + 0.9 Hz to 16.6 + 1.9Hz, with an average increase of 90.3 + 14.8% (P0.001).2. in 27 globus pallidus neurons recorded in the damage side of the 6-OHDA Parkinson's disease model rats. The micro pressure injection of 0.5mM ZD7288 could reduce the self distribution frequency of 10 (37%) neurons from 7.8 + 2.4Hz to 3.1 + 1.3Hz, with an average reduction of 70.2 + 7.. 5% (P0.01); the other 15 (55.6%) globus pallidus neurons increased to 17.1 + 3.0Hz from 7.8 + 1.4 Hz to 17.1 + 3.0Hz after micro pressure injection. The average increase was 149.8 + 29.8% (P0.001).3. in the 28 pale ball neurons recorded on the contralateral side of the rat model of 6-OHDA Parkinson's disease. The micro pressure injection of 0.5mM ZD7288 could make it possible. The spontaneous discharge frequency of the 12 (42.9%) neurons decreased from 12.8 + 2.4Hz to 7.1 1.4Hz, with an average decrease of 46.1 + 4.6% (P0.01), and the other 10 (35.7%) globus pallidus neurons increased from 7.9 + 2.4Hz to 14.3 + 3.0Hz from 7.9 + 2.4Hz to 14.3 + 3.0Hz after the micro pressure injection of 0.5mM ZD7288. The inhibitory effect of ZD7288 on the damaged side pallidus neurons was significantly enhanced than the damage to the contralateral side. The difference was statistically significant (P0.05) the mixture of.4. and D-AP5 (0.8mM) and CNQX (1.0mmM) was used to observe whether glutamate synapse transmission was involved in ZD7288 mediated excitatory effects. In 17 globus pallidus neurons, a mixture of D-AP5 and CNQX was given alone. No changes in the excitability of the globus pallidus neurons. In the presence of D-AP5 and CNQX, ZD7288 was only excited by 3 neurons (17.7%), the rate of excitatory cells was significantly lower than that of D-AP5 and CNQX (45.7%) (P0.05). In the presence of D-AP5 and CNQX, there were no significant changes in the excitability of 8 pallid neurons, and the rate of non reactive cells (47) .0%) when D-AP5 and CNQX were not given (8.6%) (8.6%) significantly increased (P0.01).5. in behavioral experiments, intraperitoneal injection of haloperidol could produce relatively stable stiffness in rats, and unilateral globus pallidus micro injection of ZD7288 could lead to different deflection behavior in Parkinson's disease rigid model rats. Among the 9 rats in the experimental group, 4 rats were displayed. The obvious contralateral deflection was statistically significant compared with that of the normal saline group (u=132.500, P0.001); the other 5 showed obvious ipsilateral deflection, compared with the normal saline group (u=9.000, P0.001).6. was observed in the white ball of normal and Parkinson's disease rats in the globus pallidus of the normal and Parkinson's disease model rats in the immunohistochemical staining experiment. 1, HCN2 and HCN4 were expressed as HCN2 subtypes, and HCN1 and HCN4 were expressed in less medium level. The three subtypes expressed.HCN1 subtypes in the cell bodies and nerve fibers of the pallid globule neurons, and the HCN2 subtypes were mainly expressed in the nerve fibers, and the cell surface was less; and HCN4 subtypes were in the neurons and nerves of the neurons. The amount of expression on the fiber is equal.
Conclusion: the results of electrophysiological study showed that ZD7288 blocking the HCN channel could change the excitability of the globus pallidus neurons in the normal and Parkinson's disease model rats. The effect of the decrease and increase of the discharge frequency was related to the transmission of glutamic acid, respectively. The behavioral experiment further revealed the microinjection of ZD in the globus pallidus by the behavioral experiment. 7288 the different deflection behavior of the Parkinson's disease rats induced by haloperidol may be related to the different electrophysiological effects of ZD7288 on the globus pallidus neurons. The morphological study showed that the expression of HCN1, HCN2 and HCN4 receptors in the globus pallidus of normal and Parkinson's disease rats.
These results provide a theoretical and experimental basis for further study of the role of globus pallidus HCN channel in the pathogenesis of Parkinson's disease.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R338

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