乙醇对小鼠小脑浦肯野细胞活动及感觉信息突触传递的影响机制

发布时间：2018-01-03 13:28

本文关键词：乙醇对小鼠小脑浦肯野细胞活动及感觉信息突触传递的影响机制　出处：《延边大学》2016年博士论文　论文类型：学位论文

【摘要】：[目的]在离体条件下,小脑浦肯野细胞对乙醇敏感,可表现为简单锋电位(SS)放电频率的改变。然而,乙醇对活体动物小脑浦肯野细胞自发性复杂锋电位(CS)的影响机制还不清楚。因此,本研究拟应用在体膜片钳记录结合药理学手段研究乙醇对乌拉坦麻醉小鼠小脑浦肯野细胞自发性CS的影响机制。[方法]成年ICR小鼠(6-8周龄)通过腹腔注射乌拉坦麻醉后,行气管插管后将动物固定在自制的脑立体定位仪上,在小脑Vermis VI-VII区相对应处行开颅手术,钻一个直径约为1-1.5mm小孔,暴露记录部位的小脑表面,用蠕动泵在脑表面持续灌流含氧的人工脑脊液(ACSF).浦肯野细胞贴附式和在体全细胞膜片钳记录使用Axopatch-200B放大器.浦肯野细胞自发性活动的全细胞记录数据通过D/A转换器1440、Clampex 10.3软件和计算机获取。记录电极内填充电极内液,填充后阻抗为4-6MΩ。在软脑膜下方150-200微米处实施浦肯野细胞全细胞记录,浦肯野细胞的认定是通过其规律的SS放电伴随有不规律CS放电特征来判断的,并通过生物素染色来确定。电生理学数据分析采用Clampfit 10.3软件,所有数据采用均数±标准误表示,并应用SPSS17.0软件进行单因素方差分析,P0.05被认为实验组之间有显著性差异。[结果](1)电流钳记录模式下,脑表面灌流乙醇(300 mM)对浦肯野细胞自发性CS频率没有显著影响,明显降低自发性CS引起的SS放电暂停时间并降低CS的AHP振幅。(2)电压钳记录模式下,乙醇(300 mM)的使用显著抑制浦肯野细胞自发性CS波型,表现为波形下面积(AUC)的减少和放电小穗数量的减少,但不改变CS自发性活动频率及其小穗即时频率。(3)乙醇导致的浦肯野细胞自发性CS波形AUC的减少具有剂量依存性,半数抑制剂量为168.5 mM。(4)阻断NMDA和mGluR1受体降低自发性CS小穗数量和AUC,但不能阻断乙醇对CS的抑制作用。(5)CB1受体阻断剂,AM251可以阻止乙醇对CS诱发SS放电暂停、AHP振幅、小穗数量和AUC的抑制作用。此外,应用CB1受体激动剂,WIN55212-2可显著抑制CS的小穗数量、AUC、CS诱发SS放电暂停和AHP振幅。[结论](1)本研究表明乙醇通过活化CB1受体导致小鼠小脑浦肯野细胞自发性CS活动抑制,提示过量饮酒可能通过活化CB1受体抑制外周感觉信息向小脑皮层浦肯野细胞的传递。(2)乙醇对自发性CS活动的影响可能在一定程度上反映急性酒精中毒对小脑皮层感觉信息传递的损伤。[目的]急性过量摄入乙醇可引起小脑运动调节功能障碍甚至共济失调。我们最近研究发现乙醇降低感觉刺激诱发小鼠小脑皮层分子层GABA能抑制性反应,提示乙醇调节面部感觉刺激在小鼠小脑浦肯野细胞上诱发的反应。因此,本研究拟应用在体膜片钳记录结合药理学手段研究乙醇对乌拉坦麻醉小鼠小脑浦肯野细胞感觉刺激诱发外向电流的影响机制。[方法]成年ICR小鼠(6-8周龄)通过腹腔注射乌拉坦麻醉后,行气管插管后将动物固定在自制的脑立体定位仪上,在小脑Vermis VI-VII区相对应处行开颅手术,钻一个直径约为1-1.5mm小孔,暴露记录部位的小脑表面,用蠕动泵在脑表面持续灌流含氧的人工脑脊液(ACSF)。浦肯野细胞贴附式和在体全细胞膜片钳记录使用Axopatch-200B放大器.浦肯野细胞自发性活动的全细胞记录数据通过D/A转换器1440、Clampex 10.3软件和计算机获取。记录电极内填充电极内液,填充后阻抗为4-6MQ。在软脑膜下方150-200微米处实施浦肯野细胞全细胞记录,浦肯野细胞的认定是通过其规律的SS放电伴随有不规律CS放电特征来判断的,并通过生物素染色来确定。应用压力喷射系统向同侧触须垫吹风(30ms,60psi)进行面部感觉刺激,吹风刺激由电脑控制并通过Master8和Clamfit10.3软件与电信号记录同步。电生理学数据分析采用Clampfit 10.3软件,所有数据采用均数±标准误表示,并应用SPSS 17.0软件进行单因素方差分析,P0.05被认为实验组之间有显著性差异。[结果](1)在电流钳记录模式下,吹风刺激小鼠触须垫可诱发小脑PC产生抑制性突触后电位(IPSP),并伴有自发性SS放电暂停。脑表面灌流乙醇(300 mM)明显抑制IPSP和自发性放电暂停时间。(2)电压钳记录模式下,乙醇(300mM)抑制感觉刺激诱发浦肯野细胞外向电流,导致外向电流振幅降低,半宽值减小,上升时间和延迟时间缩短。(3)乙醇对感觉刺激诱发小脑皮层浦肯野细胞外向电流的抑制作用具有剂量依存性,半数抑制剂量为148.5 mM。(4)应用CB1受体阻断剂,AM251和0-2050可以阻断乙醇对感觉刺激诱发小脑皮层浦肯野细胞外向电流的抑制作用。应用CB1受体激动剂也可以阻断乙醇对感觉刺激诱发小脑皮层浦肯野细胞产生外向电流的抑制作用。[结论](1)乙醇通过活化突触前CB1受体抑制感觉刺激诱发小脑皮层浦肯野细胞外向电流。(2)过量摄入乙醇对小脑皮层感觉信息的处理的损害作用有一部分是通过抑制分子层中间神经元向浦肯野细胞释放G BA引起的,这为乙醇损害小脑功能提供了一个新机制。
[Abstract]:[Objective] in vitro, cerebellar Purkinje cells are sensitive to ethanol, can be expressed as simple spike (SS) discharge frequency changes. However, ethanol on cerebellar Purkinje cells in vivo animal spontaneous complex spike (CS) the influence mechanism is not clear. Therefore, the influence mechanism. Methods this study combined with the application in vivo pharmacological means patch clamp recordings of ethanol on urethane anesthetized mouse cerebellar Purkinje cells in spontaneous CS] adult ICR mice (6-8 weeks old) by intraperitoneal injection of urethane anesthesia after tracheal intubation after the animal is fixed on the stereotaxic self-made apparatus, for craniotomy corresponding in cerebellar Vermis VI-VII region drill a hole diameter of about 1-1.5mm, exposed parts of the cerebellar surface records, continuous perfusion of oxygenated artificial cerebrospinal fluid with a peristaltic pump on the surface of the brain (ACSF). The Purkinje cell attached and in vivo whole cell membrane The use of Axopatch-200B amplifier clamp recording of spontaneous activity of Purkinje cells. Whole cell recording data through the D/A converter 1440, obtain the Clampex 10.3 software and computer. The recording electrode is filled in the pipette solution, after filling impedance is 4-6M. The implementation of Purkinje cells in the whole cell recording in the pia mater below the 150-200 micron, identification of Purkinje cells is SS through its discharge the law with irregular CS discharge characteristics to judge, and through biotin staining to determine. Electrophysiological data were analyzed by Clampfit 10.3 software, all the data by the mean and standard error, and variance analysis using SPSS17.0 software, P0.05 was considered significant. Results between the experimental groups. (1) the current clamp mode, the surface of the brain perfusion of ethanol (300 mM) had no significant effect on Purkinje cell spontaneous CS frequency significantly decreased spontaneous CS SS caused by discharge of suspended time and reduce the amplitude of AHP CS. (2) the voltage clamp mode, the use of ethanol (300 mM) significantly inhibited the spontaneous Purkinje cells showed CS wave type, area of wave (AUC) to reduce the number of spikelets and reduce discharge, but does not change the CS spontaneous activity frequency instant and spikelet frequency. (3) reduced Purkinje cell spontaneous CS waveform AUC ethanol induced with dose dependence, half inhibitor amount was 168.5 mM. (4) NMDA and mGluR1 receptor blocking AUC and reduce the number of spontaneous CS spikelet, but not block the inhibitory effect of ethanol on CS. (5) CB1 receptor antagonists AM251 can prevent the ethanol suspended on CS induced SS AHP discharge amplitude, inhibition of spikelet number and AUC. In addition, the application of CB1 receptor agonists, number of spikelets, WIN55212-2 can significantly inhibit CS induced by CS SS AUC, and AHP. Conclusion the amplitude of discharge pause] (1) this study That ethanol through the activation of CB1 receptor in mouse cerebellar Purkinje cells leads to spontaneous CS activity inhibition, suggesting that excessive drinking may be through the activation of CB1 receptor inhibits peripheral sensory information transmission to the cerebellar cortical Purkinje cells. (2) effects of ethanol on the spontaneous CS activity may reflect to a certain degree of acute alcohol intoxication on cerebellar cortical sensory information transmission injury. To excessive intake of ethanol can cause acute cerebellar ataxia and movement dysfunction. We recently found that ethanol decreased sensory stimulation of cerebellar cortex in mice induced by GABA can inhibit the reaction of the molecular layer, suggesting that ethanol modulates facial sensory evoked in mouse cerebellar Purkinje cells in the reaction. Therefore, this study combined with the application of methods in pharmacology body patch clamp recording of ethanol on urethane anesthetized mice induced by sensory stimulation of cerebellar Purkinje cells The influence mechanism of the outward current]. Methods adult ICR mice (6-8 weeks old) by intraperitoneal injection of urethane anesthesia after tracheal intubation after the animal is fixed on the stereotaxic self-made apparatus, for craniotomy corresponding in cerebellar Vermis VI-VII area, a diameter of about 1-1.5mm drill holes, exposed surface of the cerebellum the recording site, continuous perfusion of oxygenated artificial cerebrospinal fluid with a peristaltic pump on the surface of the brain (ACSF). The Purkinje cell attached and the use of Axopatch-200B amplifier in the whole cell patch clamp recording. Remember the whole cell Purkinje cell spontaneous activity recorded data through the D/A converter 1440, obtain the Clampex 10.3 software and computer recording electrodes filled. The electrode liquid filled impedance for the 4-6MQ. implementation of Purkinje cells in the whole cell recording in the pia mater below the 150-200 micron, identification of Purkinje cells through its regular discharge with no SS The regularity of CS discharge characteristics to judge, and through biotin staining. To determine the application of pressure injection system to the ipsilateral whisker pad blowing (30ms, 60psi) facial sensory stimulation, hair stimulation is controlled by computer and through Master8 and Clamfit10.3 software and record electrical synchronization. The electrophysiological data were analyzed by Clampfit 10.3 software, all the data by the mean and standard error, and SPSS 17 software was used for single factor analysis of variance, P0.05 was considered significant. Results between the experimental group (1)] in current clamp mode, blowing small stimulation can induce rat vibrissa pad of cerebellar PC produced inhibitory postsynaptic potential (IPSP), and with spontaneous discharge of SS suspension. The surface of the brain perfusion of ethanol (300 mM) significantly inhibited IPSP and spontaneous discharge pause time. (2) the voltage clamp mode, ethanol (300mM) inhibition of sensory stimulation evoked outward Purkinje cells Current, causes the amplitude of the outward current is reduced, the half width decreases, shorten the rise time and delay time. (3) inhibitory effects of ethanol on Purkinje cells in cerebellar cortex sensory evoked outward currents with dose dependence, half inhibitor amount was 148.5 mM. (4) application of CB1 receptor blockers, AM251 and 0-2050 could be blocked by ethanol the sensory evoked outward currents in Purkinje cells of cerebellar cortex inhibition. Application of CB1 receptor agonists can block the inhibition of ethanol production. Sensory evoked outward currents of Purkinje cells of the cerebellar cortex] (1) ethanol by activation of presynaptic CB1 receptors inhibits sensory evoked outward currents in cerebellar cortical Purkinje cells (2). Excessive intake of ethanol on the cerebellar cortex sensory information processing damage is part of the release of G BA to the Purkinje cell by inhibiting molecular layer interneurons Cause, this provides a new mechanism for ethanol damage of cerebellar function.

【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q42

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