【摘要】：丘腦底核(subthalamic nucleus)是基底神經(jīng)節(jié)間接環(huán)路重要的中繼核團(tuán)。有報(bào)道,其放電頻率及放電模式的改變與帕金森病直接相關(guān)。5-羥色胺(5-hydroxytryptamine,5-HT)是一種重要的單胺類(lèi)神經(jīng)遞質(zhì),在中樞神經(jīng)系統(tǒng)中發(fā)揮多種生物學(xué)效應(yīng)。有研究表明,5-HT參與帕金森病等多種神經(jīng)退行性疾病的病理生理過(guò)程。大量形態(tài)學(xué)實(shí)驗(yàn)已證實(shí),來(lái)自中縫背核的5-HT能神經(jīng)纖維可直接支配丘腦底核,并且多種5-HT受體亞型在丘腦底核神經(jīng)元均有表達(dá),其中5-HT1B、5-HT2C、5-HT4和5-HT1A受體表達(dá)較多。 目的：首先,觀察5-HT及選擇性5-HT1B、5-HT2C、5-HT4和5-HT1A受體激動(dòng)劑對(duì)正常及6-hydroxydopamine(6-OHDA)所致帕金森病模型大鼠丘腦底核神經(jīng)元放電頻率的影響。其次,比較5-HT對(duì)兩種狀態(tài)下的丘腦底核神經(jīng)元電生理效應(yīng)的異同。觀察雙側(cè)丘腦底核注射5-HT對(duì)氟哌啶醇(haloperidol)所致的大鼠僵直行為的影響。最后,為了進(jìn)一步明確5-HT電生理效應(yīng)的受體機(jī)制,觀察正常大鼠丘腦底核神經(jīng)元5-HT受體亞型5-HT1B、5-HT2C、5-HT4和5-HT1A的表達(dá)情況。 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、6-OHDA帕金森病大鼠模型制備、免疫組織化學(xué)染色及行為學(xué)等實(shí)驗(yàn)方法。 結(jié)果：1.在正常大鼠電生理實(shí)驗(yàn)中,記錄到47個(gè)丘腦底核神經(jīng)元,0.1mM5-HT可使其中32個(gè)神經(jīng)元的自發(fā)放電頻率平均升高32.02+4.31%；另外15個(gè)丘腦底核神經(jīng)元的興奮性被5-HT抑制,其放電頻率平均降低26.08±4.58%。而無(wú)菌生理鹽水對(duì)照組記錄到的8個(gè)丘腦底核神經(jīng)元,放電頻率平均升高4.89±1.61%。5-HT對(duì)丘腦底核神經(jīng)元放電頻率的升高和降低效應(yīng)分別與對(duì)照組相比,均有顯著的統(tǒng)計(jì)學(xué)意義(P0.001)。 為探討5-HT對(duì)丘腦底核神經(jīng)元興奮和抑制效應(yīng)的受體機(jī)制,在記錄到的88個(gè)丘腦底核神經(jīng)元中,分別微量加壓注射5-HTB受體激動(dòng)劑CP-9312、5-HT2C受體激動(dòng)劑RO-600175和5-HT4受體激動(dòng)劑ML-10302可使丘腦底核神經(jīng)元放電頻率升高25.11±3.79%、22.82±4.02%和23.99±2.79%,與無(wú)菌生理鹽水對(duì)照組相比均有統(tǒng)計(jì)學(xué)意義(P0.01)。而注射5-HT1A受體激動(dòng)劑8-OH-DPAT則使自發(fā)放電頻率降低18.75±2.70%,與對(duì)照組相比差異顯著(P0.01)。同時(shí),我們將5-HT對(duì)丘腦底核神經(jīng)元的興奮效應(yīng)分別與CP-93129、RO-600175和ML-10302的興奮效應(yīng)相比,無(wú)顯著差異(P0.05),而5-HT的抑制效應(yīng)與8-OH-DPAT產(chǎn)生的抑制效應(yīng)相比,也無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 為了觀察單一丘腦底核神經(jīng)元是否同時(shí)表達(dá)多種功能性5-HT受體亞型,我們對(duì)正常大鼠丘腦底核同一個(gè)神經(jīng)元先后加壓注射CP-93129和8-OH-DPAT,觀察其對(duì)放電頻率的影響。CP-93129對(duì)7個(gè)丘腦底核神經(jīng)元產(chǎn)生興奮效應(yīng),放電頻率增加23.92±7.03%(P0.05),8-OH-DAPT則使這些神經(jīng)元的基礎(chǔ)放電頻率明顯降低(降低：19.28±3.45%,P0.05)。此外,向同一個(gè)丘腦底核神經(jīng)元先后注射RO-600175和ML-10302,均使神經(jīng)元放電頻率明顯升高(RO-600175:20.04±4.40%;ML-10302:26.26±6.87%,n=11,P0.01)。 2.在帕金森病模型大鼠電生理實(shí)驗(yàn)中,損毀側(cè)記錄到的38個(gè)丘腦底核神經(jīng)元中,0.1mmM5-HT可使其中25個(gè)神經(jīng)元的自發(fā)放電頻率平均升高31.12±4.27%；另外13個(gè)丘腦底核神經(jīng)元的興奮性被5-HT抑制,放電頻率平均降低23.10±7.05%。分別微量加壓注射5-HT1B受體激動(dòng)劑CP-93129、5-HT2C受體激動(dòng)劑RO-600175和5-HT4受體激動(dòng)劑ML-10302可使丘腦底核神經(jīng)元放電頻率升高28.76-4.43%、32.50±7.12%和26.16±6.61%。而注射5-HT1A受體激動(dòng)劑8-OH-DPAT則使自發(fā)放電頻率降低18.72±3.81%。 在帕金森病模型大鼠損毀對(duì)側(cè)丘腦底核記錄到的19個(gè)神經(jīng)元中,0.1mM5-HT可使其中10個(gè)神經(jīng)元的自發(fā)放電頻率平均升高26.54±6.41%；另外9個(gè)丘腦底核神經(jīng)元的放電頻率降低21.94±5.95%。分別注射CP-93129、RO-600175和ML-10302可使丘腦底核神經(jīng)元放電頻率升高26.91±3.35%、32.75±8.72%和26.47-6.44%。而注射8-OH-DPAT則使放電頻率降低23.27±5.17%。 3.與正常大鼠(放電頻率：7.80±0.54Hz)相比,帕金森病模型大鼠損毀側(cè)丘腦底核神經(jīng)元的基礎(chǔ)放電頻率明顯升高(放電頻率：11.07±0.85Hz,P0.001)；并且放電模式總體分布也有改變(χ2=25.523,d.f.=2,P0.001),簇狀放電明顯增多(χ2=24.832,d.f=1,P0.001)而規(guī)則放電減少(χ2=8.917,d.f.=1,P0.01)。將帕金森病模型大鼠損毀側(cè)丘腦底核神經(jīng)元的基礎(chǔ)放電頻率及放電模式與損毀對(duì)側(cè)(放電頻率：7.83±0.63Hz)相比,也表現(xiàn)為損毀側(cè)神經(jīng)元放電頻率明顯升高(P0.01),且簇狀放電增多(P0.01),規(guī)則放電減少(P0.01)。 為了進(jìn)一步證實(shí)帕金森病狀態(tài)下,大鼠丘腦底核神經(jīng)元放電模式發(fā)生改變,我們對(duì)正常及帕金森病模型大鼠丘腦底核神經(jīng)元自發(fā)放電ISI的CV和FF值進(jìn)行分析,將帕金森病模型大鼠損毀側(cè)神經(jīng)元的CV值,分別與正常大鼠及未損毀側(cè)神經(jīng)元相比,損毀側(cè)神經(jīng)元的CV值明顯升高,均有統(tǒng)計(jì)學(xué)意義(正常：0.68±0.05；損毀對(duì)側(cè)：0.66±0.08；損毀側(cè)：1.00±0.09,P0.01),同時(shí)也觀察到FF值的升高,有顯著差異(正常：0.21±0.04；損毀對(duì)側(cè)：0.16±0.05；損毀側(cè)：0.39±0.08,P0.05)。 同時(shí),我們將5-HT及4種受體激動(dòng)劑對(duì)帕金森病模型大鼠損毀側(cè)丘腦底核神經(jīng)元的電生理效應(yīng)與正常大鼠相比較,結(jié)果顯示均無(wú)顯著差異(P0.05)。 4.在行為學(xué)實(shí)驗(yàn)中,大鼠腹腔注射氟哌啶醇能產(chǎn)生相對(duì)穩(wěn)定的僵直癥狀,雙側(cè)丘腦底核微量注射0.1mM5-HT(0.3μl/側(cè))能夠顯著縮短氟哌啶醇導(dǎo)致的大鼠僵直時(shí)間,與注射生理鹽水相比有顯著統(tǒng)計(jì)學(xué)意義(P0.05)。 5.利用免疫組織化學(xué)技術(shù),對(duì)正常大鼠丘腦底核神經(jīng)元5-HT1B、5-HT2C、5-HT4和5-HT1A受體進(jìn)行染色。在大鼠丘腦底核內(nèi)均觀察到5-HT1B、5-HT2C、5-HT4和5-HT1A4種受體亞型明顯的陽(yáng)性表達(dá)。 結(jié)論：電生理學(xué)研究結(jié)果顯示外源性5-HT可以改變正常大鼠和帕金森病模型大鼠丘腦底核神經(jīng)元的興奮性,產(chǎn)生使放電頻率升高和降低的雙重效應(yīng)。5-HT1B、5-HT2c和5-HT4受體被激活可以增加丘腦底核神經(jīng)元放電頻率,而5-HT1A受體的激活卻對(duì)神經(jīng)元放電頻率起抑制作用。在帕金森病狀態(tài)下,與正常大鼠相比,雖然丘腦底核神經(jīng)元基礎(chǔ)放電頻率加快且簇狀放電增多,但5-HT及相應(yīng)受體激動(dòng)劑對(duì)神經(jīng)元的生物學(xué)效應(yīng)卻沒(méi)有明顯改變。形態(tài)學(xué)研究顯示正常大鼠丘腦底核表達(dá)豐富的5-HT1B、5-HT2C、5-HT4和5-HT1A受體。 本實(shí)驗(yàn)結(jié)果為深入探討丘腦底核5-HT的生物學(xué)效應(yīng)及其在帕金森病的發(fā)生和防治中的作用提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:The subthalamic nucleus is an important relay nucleus in the indirect loop of the basal ganglia. It has been reported that the changes of its discharge frequency and pattern are directly related to Parkinson's disease. 5-hydroxytryptamine (5-HT) is an important monoamine neurotransmitter and plays a variety of biological effects in the central nervous system. A large number of morphological experiments have proved that 5-HT-ergic nerve fibers from the dorsal raphe nucleus can directly innervate the subthalamic nucleus, and a variety of 5-HT receptor subtypes are expressed in the neurons of the subthalamic nucleus, among which 5-HT1B, 5-HT2C, 5-HT4 and 5-HT1A receptors are more expressed. Many.
AIM: To observe the effects of 5-HT and selective 5-HT1B, 5-HT2C, 5-HT4 and 5-HT1A receptor agonists on the discharging frequencies of neurons in the subthalamic nucleus of normal and 6-hydroxydopamine (6-OHDA) induced Parkinson's disease rats. Secondly, to compare the electrophysiological effects of 5-HT on the neurons in the two different states. Effects of 5-HT injection on haloperidol-induced stiffness in rats. Finally, in order to further clarify the receptor mechanism of 5-HT electrophysiological effects, the expression of 5-HT receptor subtypes 5-HT1B, 5-HT2C, 5-HT4 and 5-HT1A in hypothalamic nucleus neurons of normal rats was observed.
METHODS: The in vitro electrophysiological records of multitube microelectrodes, the preparation of 6-OHDA Parkinson's disease rat model, immunohistochemical staining and behavioral experimental methods were used.
Results: 1. In normal rat electrophysiological experiment, 47 subthalamic nucleus neurons were recorded. 0.1mM5-HT could increase the spontaneous firing frequency of 32 neurons by 32.02+4.31% on average, while the excitability of 15 subthalamic nucleus neurons was inhibited by 5-HT, and the firing frequency was reduced by 26.08 +4.58% on average. The average discharge frequency of the 8 subthalamic nucleus neurons increased by 4.89 65
To investigate the receptor mechanism of excitatory and inhibitory effects of 5-HT on subthalamic nucleus neurons, microinjection of 5-HTB receptor agonist CP-9312, 5-HT2C receptor agonist RO-600175 and 5-HT4 receptor agonist ML-10302 increased the firing frequency of subthalamic nucleus neurons by 25.11 (+) 3.79%, 22.82 (+) 4. The spontaneous discharges of 5-HT1A receptor agonist 8-OH-DPAT decreased by 18.75 (+ 2.70%) compared with the control group (P 0.01). At the same time, the excitatory effects of 5-HT on neurons in the subthalamic nucleus were compared with those of CP-93129, RO-600175 and ML-103, respectively. There was no significant difference in the excitatory effect of 0.02 (P 0.05), and the inhibitory effect of 5-HT was not significantly different from that of 8-OH-DPAT (P 0.05).
To observe whether single subthalamic nucleus neurons express multiple functional 5-HT receptor subtypes at the same time, we injected CP-93129 and 8-OH-DPAT into the same neuron of the subthalamic nucleus of normal rats to observe the effect of CP-93129 on the firing frequency. In addition, RO-600175 and ML-10302 were injected successively into the same subthalamic nucleus neurons, and the firing frequency of these neurons was significantly increased (RO-600175:20.04+4.40%; ML-10302:26.26+6.87%, n=11, P 0.01).
2. In the electrophysiological experiment of Parkinson's disease model rats, 0.1 mmM5-HT could increase the spontaneous firing frequency of 25 of the 38 neurons recorded on the lesion side by 31.12 (+ 4.27%) on average, and the excitability of 13 neurons in the subthalamic nucleus was inhibited by 5-HT, which decreased by 23.10 (+ 7.05%). Radiation of 5-HT1B receptor agonist CP-93129,5-HT2C receptor agonist RO-600175 and 5-HT4 receptor agonist ML-10302 increased the firing frequency of subthalamic nucleus neurons by 28.76-4.43%, 32.50 (+ 7.12%) and 26.16 (+ 6.61%). Injection of 5-HT1A receptor agonist 8-OH-DPAT decreased the spontaneous firing frequency by 18.72 (+ 3.81%).
Of the 19 neurons recorded in the contralateral subthalamic nucleus of Parkinson's disease rats, 0.1mM5-HT increased the spontaneous firing frequency of 10 neurons by 26.54 (+ 6.41%) on average, while the firing frequency of the other 9 neurons in the subthalamic nucleus decreased by 21.94 (+ 5.95%). Injections of CP-93129, RO-600175 and ML-10302 could induce the neurons in the subthalamic nucleus. The discharging frequency increased by 26.91 (+ 3.35%), 32.75 (+ 8.72%) and 26.47 (- 6.44%) respectively, while the discharging frequency decreased by 23.27 (+ 5.17%) after injection of 8-OH-DPAT.
3. Compared with normal rats (discharge frequency: 7.80.54 Hz), the basal discharge frequency of subthalamic nucleus neurons in Parkinson's disease model rats increased significantly (discharge frequency: 11.07.85 Hz, P 0.001), and the overall distribution of discharge patterns also changed (2 = 25.523, D.F. = 2, P 0.001), and cluster discharge increased significantly (2 = 24.832, D.F = 1, P 0.001). The basal discharge frequency and pattern of the neurons in the subthalamic nucleus of Parkinson's disease rats were significantly higher than those in the contralateral side (discharge frequency: 7.83 65507 (P0.01).
In order to further confirm the changes of neuronal firing patterns in the subthalamic nucleus of rats with Parkinson's disease, we analyzed the CV and FF values of spontaneous ISI of neurons in the normal and Parkinson's disease rats, and compared the CV values of neurons in the lesioned side with those in the normal and uninjured side of neurons in Parkinson's disease rats, respectively. The CV value of the injured side was significantly higher than that of the injured side (normal: 0.68 + 0.05; contralateral: 0.66 + 0.08; injured side: 1.00 + 0.09, P 0.01), and the FF value was significantly higher (normal: 0.21 + 0.04; contralateral: 0.16 + 0.05; injured side: 0.39 + 0.08, P 0.05).
At the same time, the electrophysiological effects of 5-HT and 4 receptor agonists on neurons in the lesion of lateral subthalamic nucleus in Parkinson's disease model rats were compared with those in normal rats. The results showed that there was no significant difference (P 0.05).
4. In the behavioral experiment, haloperidol intraperitoneal injection can produce relatively stable stiffness symptoms in rats. Microinjection of 0.1mM5-HT (0.3ml/side) into bilateral subthalamic nucleus can significantly shorten the time of haloperidol-induced stiffness in rats, which is statistically significant compared with saline injection (P 0.05).
5. Immunohistochemical staining of 5-HT1B, 5-HT2C, 5-HT4 and 5-HT1A receptors in normal rat subthalamic nucleus neurons was performed.
CONCLUSION: Electrophysiological studies show that exogenous 5-HT can alter the excitability of neurons in the subthalamic nucleus of normal rats and Parkinson's disease model rats, producing a dual effect of increasing and decreasing the firing frequency. Activation of 5-HT1B, 5-HT2c and 5-HT4 receptors can increase the firing frequency of neurons in the subthalamic nucleus, whereas activation of 5-HT1A receptors can increase the firing frequency of neurons in the subthalamic nucleus In Parkinson's disease, although the basal firing frequency of thalamic subthalamic nucleus neurons was increased and the cluster firing was increased, the biological effects of 5-HT and corresponding receptor agonists on neurons were not significantly changed. Rich 5-HT1B, 5-HT2C, 5-HT4 and 5-HT1A receptors.
The results of this study provide an experimental basis for further study of the biological effects of 5-HT in the subthalamic nucleus and its role in the pathogenesis, prevention and treatment of Parkinson's disease.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：Q424;R742.5

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相關(guān)期刊論文 前1條

1 張巧俊;高蕊;劉健;劉婭萍;王爽;;帕金森病大鼠中縫背核5-羥色胺能神經(jīng)元電活動(dòng)的變化[J];生理學(xué)報(bào);2007年02期

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本文編號(hào)：2183975