正常及MPTP帕金森病模型小鼠苍白球HCN通道的电生理学研究

发布时间：2018-04-27 04:26

本文选题：苍白球 + HCN通道　；参考：《青岛大学》2014年硕士论文

【摘要】：苍白球(globus pallidus, GP)是位于基底神经节中央,参与调节多个反馈环路的中继核团,其神经纤维可投射到基底神经节几乎所有核团,在机体正常及运动障碍性疾病中发挥重要作用。研究发现超极化激活环核苷酸门控(hyperpolarization-activated cyclic nucleotide-gated, HCN)阳离子通道在苍白球神经元自发起搏放电中起重要作用。离体脑片膜片钳研究证实,阻断HCN通道明显改变苍白球神经元的起搏电活动。形态学研究显示苍白球表达有HCN通道。目的：探讨阻断HCN通道对正常小鼠及MPTP帕金森病模型小鼠苍白球神经元自发放电的影响,以及正常和帕金森病模型小鼠苍白球神经元HCN通道各亚型的表达。方法：本实验采用多管微电极在体细胞外电生理记录、MPTP帕金森病小鼠模型制备以及免疫组织化学染色等实验方法。结果：1.在体细胞外电生理实验中,共记录到77个正常小鼠苍白球神经元放电,平均自发放电频率为7.9±0.8Hz。苍白球神经元呈现三种放电模式,其中20个(25.4%)呈规则放电,43个(56.3%)呈不规则放电,14个(18.3%)呈簇状放电。2.在记录到的58个正常小鼠苍白球神经元中,有30个(51.7%)神经元在微量压力注射HCN通道阻断剂ZD7288(0.05mM)后自发放电频率由9.8±1.5Hz降低到5.0±0.9Hz,平均降低54.0±5.1%(P0.001)。放电模式分析显示,ZD7288增加CV值(加药前：0.82±0.14；加药后：1.39±0.19),差别有统计学意义(P0.001)；并使FF值由0.29±0.12增加至1.57±0.49,差别有统计学意义(P0.01)。提示ZD7288改变苍白球神经元放电模式,使规则放电减少。在另外22个(37.9%)苍白球神经元,0.05mM ZD7288可产生兴奋效应,自发放电频率由5.9±1.3Hz增加到10.8±2.1Hz,平均升高120.1±22.2%(P0.001)。ZD7288对CV值(加药前：0.91±0.11；加药后：0.90±0.12,P0.05)及FF值(加药前：0.34±0.09；加药后：0.30±0.11,P0.05)无明显影响。进一步分析显示ZD7288对放电频率的效应与苍白球神经元基础放电频率之间存在相关性,差别有统计学意义(r=-0.284,P0.05),基础放电频率低的神经元加药后较易产生兴奋效应,基础放电频率高的神经元加药后较易产生抑制效应。3.进一步实验观察了非特异性HCN通道阻断剂CsCl(1mM)对神经元自发放电的影响。在正常小鼠记录到的19个苍白球神经元中6个(31.6%)呈现抑制效应,自发放电频率由8.6±1.3Hz降低到3.9±1.1Hz,平均降低59.7±9.8%(P0.001)。另外8个(41.1%)神经元可以被CsCl兴奋,自发放电频率由4.7±1.4Hz升高至7.5±2.1Hz,平均升高81.1±17.1%(P0.05)。在正常小鼠,ZD7288和CsCl对苍白球神经元的效应无明显差异(P0.05)。4.在MPTP帕金森病模型小鼠记录到的28个苍白球神经元,平均放电频率为7.5±0.7Hz,与正常小鼠相比无明显差异(P0.05)。放电模式分析显示6个(21.4%)神经元呈规则放电,15个(53.6%)神经元呈不规则放电,7个(25.0%)神经元呈簇状放电。与正常小鼠相比,MPTP模型小鼠苍白球神经元放电模式中规则放电百分比有所减少,不规则放电和簇状放电百分比增多,但χ2检验结果显示差异无显著性(χ2=0.38,0.10,0.71d.f.=1,P0.05)；MPTP模型小鼠和正常小鼠基础放电CV值和FF值相比,差别均无统计学意义(P0.05)。5.在上述MPTP帕金森病模型小鼠记录到的28个苍白球神经元中,微量压力注射0.05mMZD7288可以使其中的10个(35.7%)神经元的自发放电频率由10.3±1.6Hz降低到5.8±1.1Hz,平均降低40.4±6.1%(P0.01)。另外15个(53.6%)苍白球神经元在微量压力注射0.05mM ZD7288后自发放电频率由7.0±1.3Hz升高到13.4±3.1Hz,平均升高150.9±53.7%(P0.05)。放电模式分析显示,上述引发兴奋和抑制效应的两组神经元加药前后CV值和FF值均无明显改变,提示ZD7288对模型小鼠苍白球神经元放电模式无明显影响。相关性分析显示,ZD7288效应与神经元基础放电频率之间存在相关性(r=-0.396,P0.05),基础放电频率低的神经元加药后较易产生兴奋效应,基础放电频率高的神经元加药后较易产生抑制效应。6.免疫组织化学染色显示小鼠苍白球表达HCN1、HCN2、HCN3和HCN4四种通道亚型。其中HCN1亚型在神经元胞体表达较多,HCN2和HCN3亚型在胞体和神经纤维表达量相当,而HCN4亚型只在神经纤维有少量表达。与正常小鼠相比,帕金森病模型小鼠苍白球HCN1、HCN2和HCN3通道亚型阳性细胞数明显减少(P0.05,P0.05,P0.001)。结论：阻断HCN通道可改变正常小鼠苍白球神经元的兴奋性,产生放电频率降低和升高两种效应,并参与调节神经元放电模式。在MPTP帕金森病模型小鼠,ZD7288仍可产生放电频率降低和升高的效应,但对神经元放电模式无明显影响。正常及帕金森病模型小鼠苍白球表达HCN1, HCN2, HCN3和HCN4通道亚型,且帕金森病模型小鼠HCN表达下调。本实验结果为进一步深入探讨苍白球HCN通道在帕金森病发病机制中的作用提供了一定的理论基础和实验依据。
[Abstract]:Globus pallidus (GP), which is located in the center of the basal ganglia, participates in the relay nuclei of multiple feedback loops, and its nerve fibers can be projected to almost all the nuclei of the basal ganglia, and play an important role in the normal and dyskinesiic diseases of the body. The hyperpolarization activated cyclic nucleotide gate control (hyperpolarization-activa) has been found. Ted cyclic nucleotide-gated, HCN) cation channels play an important role in the self initiated pacemaker discharge of pallid bulb neurons. In vitro patch clamp studies have confirmed that blocking the HCN channel significantly changes the pacing electrical activity of the pallid bulb neurons. Morphological studies show that the globus pallidus is expressed in HCN channels.
Objective: To investigate the effect of blocking HCN channel on the spontaneous discharge of globus pallidus neurons in normal mice and MPTP Parkinson's disease model mice, and the expression of HCN channel subtypes in normal and Parkinson's disease model mice.
Methods: in vitro electrophysiological recording, MPTP Parkinson disease mouse model and immunohistochemical staining were used.
Results: 1. in the external electrophysiological experiment, 77 normal mice were recorded in 77 normal mice. The average spontaneous discharge frequency was 7.9 + 0.8Hz. globus pallidus neurons in three discharge modes, of which 20 (25.4%) were regular discharge, 43 (56.3%) were irregular discharge, 14 (18.3%) showed cluster like discharge of 58 positive. In the common mouse globus pallidus neurons, 30 (51.7%) neurons were released from 9.8 + 1.5Hz to 5 + 0.9Hz after the micro pressure injection of HCN channel blocker ZD7288 (0.05mM), and the average decrease was 54 + 5.1% (P0.001). The analysis of discharge mode showed that ZD7288 increased CV value (0.82 + 0.14, 1.39 + 0.19 after adding medicine), and the difference was unified. Study significance (P0.001); and increase the value of FF from 0.29 + 0.12 to 1.57 + 0.49, the difference is statistically significant (P0.01). It suggests that ZD7288 changes the discharge mode of pallid bulb neurons and reduces the regular discharge. In the other 22 (37.9%) globus pallidus neurons, 0.05mM ZD7288 can produce an exciting effect, the spontaneous discharge frequency increases from 5.9 + 1.3Hz to 10.8 + 2.1Hz, The average increase of 120.1 + 22.2% (P0.001).ZD7288 (before adding medicine: 0.91 + 0.11; 0.90 + 0.12, P0.05) and FF value (before adding medicine: 0.34 + 0.09; 0.30 + 0.11, P0.05) had no obvious effect. Further analysis showed that the effect of ZD7288 on the frequency of discharge was correlated with the frequency of the basal discharge of globus pallidus neurons. There are statistical significance (r=-0.284, P0.05), the neurons with low basal discharge frequency are more likely to produce excitatory effect, and the neurons with high frequency of basic discharge are more likely to produce inhibition effect.3.. The effect of non specific HCN channel blocker CsCl (1mM) on the spontaneous discharge of the deity element is observed. 19 6 (31.6%) of the globus pallidus neurons showed inhibitory effect, the spontaneous discharge frequency decreased from 8.6 + 1.3Hz to 3.9 1.1Hz, and the average decrease was 59.7 + 9.8% (P0.001). The other 8 (41.1%) neurons could be excited by CsCl, the spontaneous discharge frequency increased from 4.7 + 1.4Hz to 7.5 + 2.1Hz, and the average increase was 81.1 + 17.1% (P0.05). In normal mice, ZD7288 and CsCl pairs of gray The effect of white ball neurons was not significantly different (P0.05).4. in MPTP Parkinson's disease model mice recorded 28 globus pallidus neurons, the average discharge frequency of 7.5 + 0.7Hz, compared with normal mice, no significant difference (P0.05). Discharge pattern analysis showed that 6 (21.4%) neurons were regular discharge, 15 (53.6%) neurons were irregular discharge, 7 (25) .0%) neurons were cluster discharges. Compared with normal mice, the percentage of regular discharge in the discharge mode of globus pallidus neurons in MPTP model mice decreased, and the percentage of irregular discharge and cluster discharge increased, but the result of chi 2 test showed no significant difference (x 2=0.38,0.10,0.71d.f. =1, P0.05), and MPTP model mice and normal mice foundation. There was no statistically significant difference between the CV and FF values (P0.05).5. in the 28 globus pallidus neurons recorded in the above MPTP Parkinson's disease model mice. The micro pressure injection of 0.05mMZD7288 could reduce the spontaneous discharge frequency of 10 (35.7%) neurons from 10.3 + 1.6Hz to 5.8 + 1.1Hz, with an average decrease of 40.4 + 6.1% (P0.01). The other 15. (53.6%) the spontaneous emission frequency of the globus pallidus neurons increased from 7 + 1.3Hz to 13.4 + 3.1Hz, and the average increase was 150.9 + 53.7% (P0.05) after the injection of 0.05mM ZD7288. The discharge mode analysis showed that the CV and FF values before and after the adding of the excitatory and inhibitory effects of the neurons were not significantly changed, suggesting ZD7288 to the model mice. The correlation analysis shows that there is a correlation between the ZD7288 effect and the neuronal basal discharge frequency (r=-0.396, P0.05), and the neurons with low basal discharge frequency are more likely to produce excitatory effect, and the nerve element with high basic discharge frequency is more likely to produce the inhibitory effect.6. immuno histochemistry after the addition of the nerve element with high basic discharge frequency. The coloring showed that the mouse globus pallidus expressed four channel subtypes of HCN1, HCN2, HCN3 and HCN4, in which the expression of HCN1 subtype in the cell body was more, the expression of HCN2 and HCN3 subtypes in the cell body and nerve fiber was equal, but the HCN4 subtype was only a small amount of expression in the nerve fibers. Compared with the normal mice, the globus pallidus in Parkinson's disease model mice HCN1, HCN2 and HCN3. The number of positive cells in Dao subtype decreased significantly (P0.05, P0.05, P0.001).
Conclusion: blocking the HCN channel can change the excitability of the normal mouse globus pallidus neurons, produce two effects of decreasing and increasing discharge frequency and increasing the mode of neuron discharge. In MPTP Parkinson's disease model mice, ZD7288 can still produce the effect of decreasing and increasing discharge frequency, but it has no obvious effect on the mode of neuron discharge. Expression of HCN1, HCN2, HCN3 and HCN4 channel subtypes in globus pallidus of Parkinson's disease mice, and HCN expression in mice with Parkinson's disease was down regulated.
The results of this study provide a theoretical basis and experimental evidence for further exploring 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】：2014
【分类号】：R742.5

【共引文献】