促肾上腺皮质激素释放激素对小鼠小脑皮层浦肯野细胞简单峰电位活动的影响

发布时间：2018-01-14 20:34

  本文关键词：促肾上腺皮质激素释放激素对小鼠小脑皮层浦肯野细胞简单峰电位活动的影响　出处：《延边大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 小脑皮层 促肾上腺皮质激素释放激素 细胞贴附式记录 小脑浦肯野细胞 神经药理学

【摘要】：[目的]小脑是哺乳动物重要的运动中枢,外部信息经攀爬纤维(Climbing fiber,CF)和苔藓纤维(Mossy fiber,MF)传入小脑皮层,经精密运算后,由浦肯野细胞(Purkinje cell,PC)发出各种输出指令,来完成各种生理活动。促肾上腺皮质激素释放激素(Corticotropin releasing hormone,CRH)是调控应激反应和内脏活动的一种重要神经肽,CRH可通过传入纤维分布于哺乳动物小脑环路,影响PCs的自发性放电和指令输出,但其影响机制尚不明确。本研究拟采用在体的电生理记录技术以及神经药理学手段,在乌拉坦麻醉的状态下,研究不同浓度CRH对小鼠小脑PC自发性单纯峰电位活动的影响,探讨CRH对小脑皮层PC自发性活动影响的受体机制。[实验材料与方法]成年(6-8周龄)ICR小鼠,腹腔注射乌拉坦(1.3 g/kg)麻醉后,于小脑Vermis区的相应部位行一个直径为1-1.5 mm的开颅术,剥除硬脑膜,脑表面的暴露部位持续给予人工脑脊液(Artificial cerebrospinal fluid,ACSF)灌流。PC自发性放电活动采用细胞外贴附式记录记录方法,通过膜片钳放大器(AxopatCh--200B)及数据采集软件记录小脑皮层PC自发性活动;CRH及受体阻断剂采用小脑表面灌流给药;电生理实验数据分析采用Clampfit 10.3软件。各组数据均用Mean 土 S.E.M.表示,记录细胞数用n表示.采用SPSS(Version 21)软件进行数据统计学的分析,使用单因素方差分析进行均数的比较,P0.05认为有统计学意义。[结果](1)脑表面灌流CRH,可导致PC自发性简单峰电位放电即时频率显著减少,峰值左移;简单峰电位放电频率由给药前的24.3 ± 1.6 Hz降低到19.4 ± 2.1 Hz(P0.05),但CRF对简单峰电位放电的变异系数(Coefficient of variation,CV)无显著影响。(2)在GABAA受体阻断剂存在下,CRH可导致PC自发性简单峰电位放电即时频率显著增加,峰值右移。简单峰电位放电频率由25.5 ± 1.8 Hz升高到30.7 ±2.4 Hz(P0.05),表明CRF可能兴奋小脑皮层抑制性中间神经元,导致PC简单峰电位放电频率减少;阻断抑制性中间神经元的抑制性传入后,CRH可兴奋PC导致简单峰电位放电频率升高。(3.)在阻断抑制性中间神经元介导的GABA能突触传递后,CRH对PC自发性简单峰电位放电频率的升高作用具有浓度依存性,引起PC自发性简单峰电位升高的半数有效浓度是53.8 nM,最大浓度是1μ M。(4)阻断GABAA和AMPA受体,CRH可使PC自发性简单峰电位频率升高,即时频率显著升高,峰值右移;CRH对fPC自发性简单峰电位放电CV影响不大,表明CRH兴奋PC导致简单峰电位放电频率升高不是通过增加CF和PF的兴奋性传入来实现的。(5)在非选择性CRH受体阻断剂存在下,CRH对PC自发性简单峰电位放电即时频率影响消失,简单峰电位平均频率不再升高,表明CRH通过CRF受体兴奋小脑皮层PC,导致PC自发性简单峰电位放电频率升高。(6)在CRH-R2阻断剂存在下,CRH对PC自发性简单峰电位放电即时频率的影响依然存在,简单峰电位平均频率显著升高,表明CRH兴奋小脑皮层PC,不依赖于CRH-R2,提示CRH直接作用于PC膜上的CRH-R1而兴奋PC导致自发性简单峰电位频率升高。[结论]本研究结果表明,CRH可兴奋小脑皮层抑制性中间神经元导致PC抑制,阻断MLI-PC间的GABA能突触传递后,CRH通过CRH-R1兴奋PC,导致PC自发性简单峰电位放电频率升高,提示应激刺激可通过CRH受体作用于小脑皮层PC,影响PC的指令输出以及运动调节功能。
[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.

【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R338
，

本文编号：1425204