本文關(guān)鍵詞：促腎上腺皮質(zhì)激素釋放激素對小鼠小腦皮層浦肯野細(xì)胞簡單峰電位活動的影響　出處：《延邊大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 小腦皮層 促腎上腺皮質(zhì)激素釋放激素 細(xì)胞貼附式記錄 小腦浦肯野細(xì)胞 神經(jīng)藥理學(xué)

【摘要】：[目的]小腦是哺乳動物重要的運(yùn)動中樞,外部信息經(jīng)攀爬纖維(Climbing fiber,CF)和苔蘚纖維(Mossy fiber,MF)傳入小腦皮層,經(jīng)精密運(yùn)算后,由浦肯野細(xì)胞(Purkinje cell,PC)發(fā)出各種輸出指令,來完成各種生理活動。促腎上腺皮質(zhì)激素釋放激素(Corticotropin releasing hormone,CRH)是調(diào)控應(yīng)激反應(yīng)和內(nèi)臟活動的一種重要神經(jīng)肽,CRH可通過傳入纖維分布于哺乳動物小腦環(huán)路,影響PCs的自發(fā)性放電和指令輸出,但其影響機(jī)制尚不明確。本研究擬采用在體的電生理記錄技術(shù)以及神經(jīng)藥理學(xué)手段,在烏拉坦麻醉的狀態(tài)下,研究不同濃度CRH對小鼠小腦PC自發(fā)性單純峰電位活動的影響,探討CRH對小腦皮層PC自發(fā)性活動影響的受體機(jī)制。[實(shí)驗(yàn)材料與方法]成年(6-8周齡)ICR小鼠,腹腔注射烏拉坦(1.3 g/kg)麻醉后,于小腦Vermis區(qū)的相應(yīng)部位行一個直徑為1-1.5 mm的開顱術(shù),剝除硬腦膜,腦表面的暴露部位持續(xù)給予人工腦脊液(Artificial cerebrospinal fluid,ACSF)灌流。PC自發(fā)性放電活動采用細(xì)胞外貼附式記錄記錄方法,通過膜片鉗放大器(AxopatCh--200B)及數(shù)據(jù)采集軟件記錄小腦皮層PC自發(fā)性活動;CRH及受體阻斷劑采用小腦表面灌流給藥;電生理實(shí)驗(yàn)數(shù)據(jù)分析采用Clampfit 10.3軟件。各組數(shù)據(jù)均用Mean 土 S.E.M.表示,記錄細(xì)胞數(shù)用n表示.采用SPSS(Version 21)軟件進(jìn)行數(shù)據(jù)統(tǒng)計學(xué)的分析,使用單因素方差分析進(jìn)行均數(shù)的比較,P0.05認(rèn)為有統(tǒng)計學(xué)意義。[結(jié)果](1)腦表面灌流CRH,可導(dǎo)致PC自發(fā)性簡單峰電位放電即時頻率顯著減少,峰值左移;簡單峰電位放電頻率由給藥前的24.3 ± 1.6 Hz降低到19.4 ± 2.1 Hz(P0.05),但CRF對簡單峰電位放電的變異系數(shù)(Coefficient of variation,CV)無顯著影響。(2)在GABAA受體阻斷劑存在下,CRH可導(dǎo)致PC自發(fā)性簡單峰電位放電即時頻率顯著增加,峰值右移。簡單峰電位放電頻率由25.5 ± 1.8 Hz升高到30.7 ±2.4 Hz(P0.05),表明CRF可能興奮小腦皮層抑制性中間神經(jīng)元,導(dǎo)致PC簡單峰電位放電頻率減少;阻斷抑制性中間神經(jīng)元的抑制性傳入后,CRH可興奮PC導(dǎo)致簡單峰電位放電頻率升高。(3.)在阻斷抑制性中間神經(jīng)元介導(dǎo)的GABA能突觸傳遞后,CRH對PC自發(fā)性簡單峰電位放電頻率的升高作用具有濃度依存性,引起PC自發(fā)性簡單峰電位升高的半數(shù)有效濃度是53.8 nM,最大濃度是1μ M。(4)阻斷GABAA和AMPA受體,CRH可使PC自發(fā)性簡單峰電位頻率升高,即時頻率顯著升高,峰值右移;CRH對fPC自發(fā)性簡單峰電位放電CV影響不大,表明CRH興奮PC導(dǎo)致簡單峰電位放電頻率升高不是通過增加CF和PF的興奮性傳入來實(shí)現(xiàn)的。(5)在非選擇性CRH受體阻斷劑存在下,CRH對PC自發(fā)性簡單峰電位放電即時頻率影響消失,簡單峰電位平均頻率不再升高,表明CRH通過CRF受體興奮小腦皮層PC,導(dǎo)致PC自發(fā)性簡單峰電位放電頻率升高。(6)在CRH-R2阻斷劑存在下,CRH對PC自發(fā)性簡單峰電位放電即時頻率的影響依然存在,簡單峰電位平均頻率顯著升高,表明CRH興奮小腦皮層PC,不依賴于CRH-R2,提示CRH直接作用于PC膜上的CRH-R1而興奮PC導(dǎo)致自發(fā)性簡單峰電位頻率升高。[結(jié)論]本研究結(jié)果表明,CRH可興奮小腦皮層抑制性中間神經(jīng)元導(dǎo)致PC抑制,阻斷MLI-PC間的GABA能突觸傳遞后,CRH通過CRH-R1興奮PC,導(dǎo)致PC自發(fā)性簡單峰電位放電頻率升高,提示應(yīng)激刺激可通過CRH受體作用于小腦皮層PC,影響PC的指令輸出以及運(yùn)動調(diào)節(jié)功能。
[Abstract]:[Objective] is an important hub of mammalian cerebellar movement, external information by climbing fiber (Climbing fiber, CF) and mossy fiber (Mossy fiber MF) was introduced into the cerebellar cortex, the precise operation by Purkinje cells (Purkinje, cell, PC) issued a variety of output instructions, to complete all kinds of physiological activities. Adrenocorticotropic hormone the release of the hormone (Corticotropin releasing hormone, CRH) is an important neuropeptide regulation of stress and visceral activity, CRH by afferent fibers distributed in mammalian cerebellar circuits, PCs spontaneous discharge and command output, but its influence mechanism is not clear. This study used in vivo electrophysiological recording techniques and nerve means of pharmacology in urethane anesthetized condition, effects of different concentrations of CRH on mouse cerebellar PC spontaneous simple peak potential activities, to discuss the effects of CRH on spontaneous activity of cerebellar cortex PC Experimental materials and methods. The influence of the receptor mechanism of adults (6-8 weeks) ICR mice were intraperitoneally injected with urethane (1.3 g/kg) after anesthesia, the corresponding parts in the cerebellar Vermis area of a 1-1.5 diameter mm craniotomy, stripping the dura, exposed parts of the brain surface for artificial cerebrospinal fluid (Artificial cerebrospinal fluid, ACSF) perfusion.PC spontaneous discharge activity by extracellular recording method attached, by patch clamp amplifier (AxopatCh--200B) and data acquisition software to record the cerebellar cortex PC and spontaneous activity; CRH receptor antagonist of the cerebellar surface perfusion administration; electrophysiological experimental data analysis using Clampfit 10.3 software. All data were expressed Mean soil S.E.M., cell numbers were recorded with n. Using SPSS (Version 21) analysis software for data statistics and analysis were the number of single factor variance, P0.05 There was statistical significance. Results: (1) the surface of the brain perfusion CRH, can lead to spontaneous PC simple spike discharge instant frequency was significantly reduced, the peak shifts to the left; simple spike discharge frequency by pre administration of 24.3 + 1.6 Hz reduced to 19.4 + 2.1 Hz (P0.05), but the CRF of simple spike discharge the coefficient of variation (Coefficient of, variation, CV) had no significant effect. (2) in the presence of GABAA receptor antagonist, CRH PC can lead to spontaneous simple spike discharge instant frequency increased significantly, the peak shift. Simple spike discharge frequency from 25.5 + 1.8 Hz increased to 30.7 + 2.4 Hz (P0.05), showed that CRF may be excited cerebellar cortical inhibitory interneurons, leading to PC simple spike discharge frequency decrease; blocking inhibitory interneuron afferent, CRH excited PC leads to simple spike discharge frequency increased. (3.) in the blocking inhibitory interneurons mediated GABA synaptic transmission, increased CRH PC on spontaneous simple spike discharge frequency with concentration dependence caused by Iabour PC spontaneous simple peak potential increase is 53.8 nM, the maximum concentration is 1 M. (4) GABAA and blocking AMPA receptor, CRH can make PC spontaneous single spike frequency increases the instant frequency increases, the peak shift of fPC CRH; spontaneous simple spike discharge has little effect on the CV, showed that CRH excited PC leads to simple spike discharge frequency was not realized by increasing CF and PF afferent excitability. (5) in the non selective CRH receptor antagonist CRH the presence of PC simple spontaneous disappearance the peak potential discharge instantaneous frequency, the mean frequency of single spike is no longer increasing, that CRH through CRF receptor activation in cerebellar cortex PC, PC leads to spontaneous simple spike discharge frequency increases. (6) in the presence of CRH-R2 blocking agent Under the influence of CRH on spontaneous PC simple spike discharge instantaneous frequency still exists, the simple average frequency peak potential was significantly increased, the cerebellar cortex showed that CRH excited PC, not dependent on CRH-R2, suggesting that CRH acts directly on PC membrane CRH-R1 and PC excited cause spontaneous potential peak frequency increases. The simple conclusion] the results of this study show that CRH can excite cerebellar cortical inhibitory interneurons leads to inhibition of PC, blocking MLI-PC between GABA synaptic transmission, CRH excited by CRH-R1 PC PC, lead to spontaneous simple spike discharge frequency increased, suggesting that stress stimulation by CRH receptor in cerebellar cortex PC, affect PC command output and motion control function.

【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R338
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本文編號：1425204