本文選題：蒼白球 + HCN通道��； 參考：《青島大學(xué)》2014年碩士論文

【摘要】：蒼白球(globus pallidus, GP)是位于基底神經(jīng)節(jié)中央,參與調(diào)節(jié)多個(gè)反饋環(huán)路的中繼核團(tuán),其神經(jīng)纖維可投射到基底神經(jīng)節(jié)幾乎所有核團(tuán),在機(jī)體正常及運(yùn)動(dòng)障礙性疾病中發(fā)揮重要作用。研究發(fā)現(xiàn)超極化激活環(huán)核苷酸門控(hyperpolarization-activated cyclic nucleotide-gated, HCN)陽(yáng)離子通道在蒼白球神經(jīng)元自發(fā)起搏放電中起重要作用。離體腦片膜片鉗研究證實(shí),阻斷HCN通道明顯改變蒼白球神經(jīng)元的起搏電活動(dòng)。形態(tài)學(xué)研究顯示蒼白球表達(dá)有HCN通道。 目的：探討阻斷HCN通道對(duì)正常小鼠及MPTP帕金森病模型小鼠蒼白球神經(jīng)元自發(fā)放電的影響,以及正常和帕金森病模型小鼠蒼白球神經(jīng)元HCN通道各亞型的表達(dá)。 方法：本實(shí)驗(yàn)采用多管微電極在體細(xì)胞外電生理記錄、MPTP帕金森病小鼠模型制備以及免疫組織化學(xué)染色等實(shí)驗(yàn)方法。 結(jié)果：1.在體細(xì)胞外電生理實(shí)驗(yàn)中,共記錄到77個(gè)正常小鼠蒼白球神經(jīng)元放電,平均自發(fā)放電頻率為7.9±0.8Hz。蒼白球神經(jīng)元呈現(xiàn)三種放電模式,其中20個(gè)(25.4%)呈規(guī)則放電,43個(gè)(56.3%)呈不規(guī)則放電,14個(gè)(18.3%)呈簇狀放電。2.在記錄到的58個(gè)正常小鼠蒼白球神經(jīng)元中,有30個(gè)(51.7%)神經(jīng)元在微量壓力注射HCN通道阻斷劑ZD7288(0.05mM)后自發(fā)放電頻率由9.8±1.5Hz降低到5.0±0.9Hz,平均降低54.0±5.1%(P0.001)。放電模式分析顯示,ZD7288增加CV值(加藥前：0.82±0.14；加藥后：1.39±0.19),差別有統(tǒng)計(jì)學(xué)意義(P0.001)；并使FF值由0.29±0.12增加至1.57±0.49,差別有統(tǒng)計(jì)學(xué)意義(P0.01)。提示ZD7288改變蒼白球神經(jīng)元放電模式,使規(guī)則放電減少。在另外22個(gè)(37.9%)蒼白球神經(jīng)元,0.05mM ZD7288可產(chǎn)生興奮效應(yīng),自發(fā)放電頻率由5.9±1.3Hz增加到10.8±2.1Hz,平均升高120.1±22.2%(P0.001)。ZD7288對(duì)CV值(加藥前：0.91±0.11；加藥后：0.90±0.12,P0.05)及FF值(加藥前：0.34±0.09；加藥后：0.30±0.11,P0.05)無(wú)明顯影響。進(jìn)一步分析顯示ZD7288對(duì)放電頻率的效應(yīng)與蒼白球神經(jīng)元基礎(chǔ)放電頻率之間存在相關(guān)性,差別有統(tǒng)計(jì)學(xué)意義(r=-0.284,P0.05),基礎(chǔ)放電頻率低的神經(jīng)元加藥后較易產(chǎn)生興奮效應(yīng),基礎(chǔ)放電頻率高的神經(jīng)元加藥后較易產(chǎn)生抑制效應(yīng)。3.進(jìn)一步實(shí)驗(yàn)觀察了非特異性HCN通道阻斷劑CsCl(1mM)對(duì)神經(jīng)元自發(fā)放電的影響。在正常小鼠記錄到的19個(gè)蒼白球神經(jīng)元中6個(gè)(31.6%)呈現(xiàn)抑制效應(yīng),自發(fā)放電頻率由8.6±1.3Hz降低到3.9±1.1Hz,平均降低59.7±9.8%(P0.001)。另外8個(gè)(41.1%)神經(jīng)元可以被CsCl興奮,自發(fā)放電頻率由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對(duì)蒼白球神經(jīng)元的效應(yīng)無(wú)明顯差異(P0.05)。4.在MPTP帕金森病模型小鼠記錄到的28個(gè)蒼白球神經(jīng)元,平均放電頻率為7.5±0.7Hz,與正常小鼠相比無(wú)明顯差異(P0.05)。放電模式分析顯示6個(gè)(21.4%)神經(jīng)元呈規(guī)則放電,15個(gè)(53.6%)神經(jīng)元呈不規(guī)則放電,7個(gè)(25.0%)神經(jīng)元呈簇狀放電。與正常小鼠相比,MPTP模型小鼠蒼白球神經(jīng)元放電模式中規(guī)則放電百分比有所減少,不規(guī)則放電和簇狀放電百分比增多,但χ2檢驗(yàn)結(jié)果顯示差異無(wú)顯著性(χ2=0.38,0.10,0.71d.f.=1,P0.05)；MPTP模型小鼠和正常小鼠基礎(chǔ)放電CV值和FF值相比,差別均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。5.在上述MPTP帕金森病模型小鼠記錄到的28個(gè)蒼白球神經(jīng)元中,微量壓力注射0.05mMZD7288可以使其中的10個(gè)(35.7%)神經(jīng)元的自發(fā)放電頻率由10.3±1.6Hz降低到5.8±1.1Hz,平均降低40.4±6.1%(P0.01)。另外15個(gè)(53.6%)蒼白球神經(jīng)元在微量壓力注射0.05mM ZD7288后自發(fā)放電頻率由7.0±1.3Hz升高到13.4±3.1Hz,平均升高150.9±53.7%(P0.05)。放電模式分析顯示,上述引發(fā)興奮和抑制效應(yīng)的兩組神經(jīng)元加藥前后CV值和FF值均無(wú)明顯改變,提示ZD7288對(duì)模型小鼠蒼白球神經(jīng)元放電模式無(wú)明顯影響。相關(guān)性分析顯示,ZD7288效應(yīng)與神經(jīng)元基礎(chǔ)放電頻率之間存在相關(guān)性(r=-0.396,P0.05),基礎(chǔ)放電頻率低的神經(jīng)元加藥后較易產(chǎn)生興奮效應(yīng),基礎(chǔ)放電頻率高的神經(jīng)元加藥后較易產(chǎn)生抑制效應(yīng)。6.免疫組織化學(xué)染色顯示小鼠蒼白球表達(dá)HCN1、HCN2、HCN3和HCN4四種通道亞型。其中HCN1亞型在神經(jīng)元胞體表達(dá)較多,HCN2和HCN3亞型在胞體和神經(jīng)纖維表達(dá)量相當(dāng),而HCN4亞型只在神經(jīng)纖維有少量表達(dá)。與正常小鼠相比,帕金森病模型小鼠蒼白球HCN1、HCN2和HCN3通道亞型陽(yáng)性細(xì)胞數(shù)明顯減少(P0.05,P0.05,P0.001)。 結(jié)論：阻斷HCN通道可改變正常小鼠蒼白球神經(jīng)元的興奮性,產(chǎn)生放電頻率降低和升高兩種效應(yīng),并參與調(diào)節(jié)神經(jīng)元放電模式。在MPTP帕金森病模型小鼠,ZD7288仍可產(chǎn)生放電頻率降低和升高的效應(yīng),但對(duì)神經(jīng)元放電模式無(wú)明顯影響。正常及帕金森病模型小鼠蒼白球表達(dá)HCN1, HCN2, HCN3和HCN4通道亞型,且帕金森病模型小鼠HCN表達(dá)下調(diào)。 本實(shí)驗(yàn)結(jié)果為進(jìn)一步深入探討蒼白球HCN通道在帕金森病發(fā)病機(jī)制中的作用提供了一定的理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。
[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.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.5

【共引文獻(xiàn)】

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