本文關(guān)鍵詞：乙醇對小鼠小腦浦肯野細(xì)胞活動及感覺信息突觸傳遞的影響機(jī)制　出處：《延邊大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 小腦浦肯野細(xì)胞 復(fù)雜鋒電位 簡單鋒電位 CB1受體 在體膜片鉗記錄 乙醇 乙醇 小腦皮層浦肯野細(xì)胞 分子層中間神經(jīng)元 感覺刺激 外向電流 CB1受體 在體膜片鉗記錄 神經(jīng)藥理學(xué)

【摘要】：[目的]在離體條件下,小腦浦肯野細(xì)胞對乙醇敏感,可表現(xiàn)為簡單鋒電位(SS)放電頻率的改變。然而,乙醇對活體動物小腦浦肯野細(xì)胞自發(fā)性復(fù)雜鋒電位(CS)的影響機(jī)制還不清楚。因此,本研究擬應(yīng)用在體膜片鉗記錄結(jié)合藥理學(xué)手段研究乙醇對烏拉坦麻醉小鼠小腦浦肯野細(xì)胞自發(fā)性CS的影響機(jī)制。[方法]成年ICR小鼠(6-8周齡)通過腹腔注射烏拉坦麻醉后,行氣管插管后將動物固定在自制的腦立體定位儀上,在小腦Vermis VI-VII區(qū)相對應(yīng)處行開顱手術(shù),鉆一個直徑約為1-1.5mm小孔,暴露記錄部位的小腦表面,用蠕動泵在腦表面持續(xù)灌流含氧的人工腦脊液(ACSF).浦肯野細(xì)胞貼附式和在體全細(xì)胞膜片鉗記錄使用Axopatch-200B放大器.浦肯野細(xì)胞自發(fā)性活動的全細(xì)胞記錄數(shù)據(jù)通過D/A轉(zhuǎn)換器1440、Clampex 10.3軟件和計算機(jī)獲取。記錄電極內(nèi)填充電極內(nèi)液,填充后阻抗為4-6MΩ。在軟腦膜下方150-200微米處實(shí)施浦肯野細(xì)胞全細(xì)胞記錄,浦肯野細(xì)胞的認(rèn)定是通過其規(guī)律的SS放電伴隨有不規(guī)律CS放電特征來判斷的,并通過生物素染色來確定。電生理學(xué)數(shù)據(jù)分析采用Clampfit 10.3軟件,所有數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)誤表示,并應(yīng)用SPSS17.0軟件進(jìn)行單因素方差分析,P0.05被認(rèn)為實(shí)驗(yàn)組之間有顯著性差異。[結(jié)果](1)電流鉗記錄模式下,腦表面灌流乙醇(300 mM)對浦肯野細(xì)胞自發(fā)性CS頻率沒有顯著影響,明顯降低自發(fā)性CS引起的SS放電暫停時間并降低CS的AHP振幅。(2)電壓鉗記錄模式下,乙醇(300 mM)的使用顯著抑制浦肯野細(xì)胞自發(fā)性CS波型,表現(xiàn)為波形下面積(AUC)的減少和放電小穗數(shù)量的減少,但不改變CS自發(fā)性活動頻率及其小穗即時頻率。(3)乙醇導(dǎo)致的浦肯野細(xì)胞自發(fā)性CS波形AUC的減少具有劑量依存性,半數(shù)抑制劑量為168.5 mM。(4)阻斷NMDA和mGluR1受體降低自發(fā)性CS小穗數(shù)量和AUC,但不能阻斷乙醇對CS的抑制作用。(5)CB1受體阻斷劑,AM251可以阻止乙醇對CS誘發(fā)SS放電暫停、AHP振幅、小穗數(shù)量和AUC的抑制作用。此外,應(yīng)用CB1受體激動劑,WIN55212-2可顯著抑制CS的小穗數(shù)量、AUC、CS誘發(fā)SS放電暫停和AHP振幅。[結(jié)論](1)本研究表明乙醇通過活化CB1受體導(dǎo)致小鼠小腦浦肯野細(xì)胞自發(fā)性CS活動抑制,提示過量飲酒可能通過活化CB1受體抑制外周感覺信息向小腦皮層浦肯野細(xì)胞的傳遞。(2)乙醇對自發(fā)性CS活動的影響可能在一定程度上反映急性酒精中毒對小腦皮層感覺信息傳遞的損傷。[目的]急性過量攝入乙醇可引起小腦運(yùn)動調(diào)節(jié)功能障礙甚至共濟(jì)失調(diào)。我們最近研究發(fā)現(xiàn)乙醇降低感覺刺激誘發(fā)小鼠小腦皮層分子層GABA能抑制性反應(yīng),提示乙醇調(diào)節(jié)面部感覺刺激在小鼠小腦浦肯野細(xì)胞上誘發(fā)的反應(yīng)。因此,本研究擬應(yīng)用在體膜片鉗記錄結(jié)合藥理學(xué)手段研究乙醇對烏拉坦麻醉小鼠小腦浦肯野細(xì)胞感覺刺激誘發(fā)外向電流的影響機(jī)制。[方法]成年ICR小鼠(6-8周齡)通過腹腔注射烏拉坦麻醉后,行氣管插管后將動物固定在自制的腦立體定位儀上,在小腦Vermis VI-VII區(qū)相對應(yīng)處行開顱手術(shù),鉆一個直徑約為1-1.5mm小孔,暴露記錄部位的小腦表面,用蠕動泵在腦表面持續(xù)灌流含氧的人工腦脊液(ACSF)。浦肯野細(xì)胞貼附式和在體全細(xì)胞膜片鉗記錄使用Axopatch-200B放大器.浦肯野細(xì)胞自發(fā)性活動的全細(xì)胞記錄數(shù)據(jù)通過D/A轉(zhuǎn)換器1440、Clampex 10.3軟件和計算機(jī)獲取。記錄電極內(nèi)填充電極內(nèi)液,填充后阻抗為4-6MQ。在軟腦膜下方150-200微米處實(shí)施浦肯野細(xì)胞全細(xì)胞記錄,浦肯野細(xì)胞的認(rèn)定是通過其規(guī)律的SS放電伴隨有不規(guī)律CS放電特征來判斷的,并通過生物素染色來確定。應(yīng)用壓力噴射系統(tǒng)向同側(cè)觸須墊吹風(fēng)(30ms,60psi)進(jìn)行面部感覺刺激,吹風(fēng)刺激由電腦控制并通過Master8和Clamfit10.3軟件與電信號記錄同步。電生理學(xué)數(shù)據(jù)分析采用Clampfit 10.3軟件,所有數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)誤表示,并應(yīng)用SPSS 17.0軟件進(jìn)行單因素方差分析,P0.05被認(rèn)為實(shí)驗(yàn)組之間有顯著性差異。[結(jié)果](1)在電流鉗記錄模式下,吹風(fēng)刺激小鼠觸須墊可誘發(fā)小腦PC產(chǎn)生抑制性突觸后電位(IPSP),并伴有自發(fā)性SS放電暫停。腦表面灌流乙醇(300 mM)明顯抑制IPSP和自發(fā)性放電暫停時間。(2)電壓鉗記錄模式下,乙醇(300mM)抑制感覺刺激誘發(fā)浦肯野細(xì)胞外向電流,導(dǎo)致外向電流振幅降低,半寬值減小,上升時間和延遲時間縮短。(3)乙醇對感覺刺激誘發(fā)小腦皮層浦肯野細(xì)胞外向電流的抑制作用具有劑量依存性,半數(shù)抑制劑量為148.5 mM。(4)應(yīng)用CB1受體阻斷劑,AM251和0-2050可以阻斷乙醇對感覺刺激誘發(fā)小腦皮層浦肯野細(xì)胞外向電流的抑制作用。應(yīng)用CB1受體激動劑也可以阻斷乙醇對感覺刺激誘發(fā)小腦皮層浦肯野細(xì)胞產(chǎn)生外向電流的抑制作用。[結(jié)論](1)乙醇通過活化突觸前CB1受體抑制感覺刺激誘發(fā)小腦皮層浦肯野細(xì)胞外向電流。(2)過量攝入乙醇對小腦皮層感覺信息的處理的損害作用有一部分是通過抑制分子層中間神經(jīng)元向浦肯野細(xì)胞釋放G BA引起的,這為乙醇損害小腦功能提供了一個新機(jī)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.

【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q42

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