正常及帕金森病模型大鼠丘脑底核5-羟色胺的电生理效应及其受体机制研究
发布时间：2018-08-14 20:05
【摘要】：丘脑底核(subthalamic nucleus)是基底神经节间接环路重要的中继核团。有报道,其放电频率及放电模式的改变与帕金森病直接相关。5-羟色胺(5-hydroxytryptamine,5-HT)是一种重要的单胺类神经递质,在中枢神经系统中发挥多种生物学效应。有研究表明,5-HT参与帕金森病等多种神经退行性疾病的病理生理过程。大量形态学实验已证实,来自中缝背核的5-HT能神经纤维可直接支配丘脑底核,并且多种5-HT受体亚型在丘脑底核神经元均有表达,其中5-HT1B、5-HT2C、5-HT4和5-HT1A受体表达较多。 目的：首先,观察5-HT及选择性5-HT1B、5-HT2C、5-HT4和5-HT1A受体激动剂对正常及6-hydroxydopamine(6-OHDA)所致帕金森病模型大鼠丘脑底核神经元放电频率的影响。其次,比较5-HT对两种状态下的丘脑底核神经元电生理效应的异同。观察双侧丘脑底核注射5-HT对氟哌啶醇(haloperidol)所致的大鼠僵直行为的影响。最后,为了进一步明确5-HT电生理效应的受体机制,观察正常大鼠丘脑底核神经元5-HT受体亚型5-HT1B、5-HT2C、5-HT4和5-HT1A的表达情况。 方法：本实验采用多管微电极在体细胞外电生理记录、6-OHDA帕金森病大鼠模型制备、免疫组织化学染色及行为学等实验方法。 结果：1.在正常大鼠电生理实验中,记录到47个丘脑底核神经元,0.1mM5-HT可使其中32个神经元的自发放电频率平均升高32.02+4.31%；另外15个丘脑底核神经元的兴奋性被5-HT抑制,其放电频率平均降低26.08±4.58%。而无菌生理盐水对照组记录到的8个丘脑底核神经元,放电频率平均升高4.89±1.61%。5-HT对丘脑底核神经元放电频率的升高和降低效应分别与对照组相比,均有显著的统计学意义(P0.001)。 为探讨5-HT对丘脑底核神经元兴奋和抑制效应的受体机制,在记录到的88个丘脑底核神经元中,分别微量加压注射5-HTB受体激动剂CP-9312、5-HT2C受体激动剂RO-600175和5-HT4受体激动剂ML-10302可使丘脑底核神经元放电频率升高25.11±3.79%、22.82±4.02%和23.99±2.79%,与无菌生理盐水对照组相比均有统计学意义(P0.01)。而注射5-HT1A受体激动剂8-OH-DPAT则使自发放电频率降低18.75±2.70%,与对照组相比差异显著(P0.01)。同时,我们将5-HT对丘脑底核神经元的兴奋效应分别与CP-93129、RO-600175和ML-10302的兴奋效应相比,无显著差异(P0.05),而5-HT的抑制效应与8-OH-DPAT产生的抑制效应相比,也无统计学意义(P0.05)。 为了观察单一丘脑底核神经元是否同时表达多种功能性5-HT受体亚型,我们对正常大鼠丘脑底核同一个神经元先后加压注射CP-93129和8-OH-DPAT,观察其对放电频率的影响。CP-93129对7个丘脑底核神经元产生兴奋效应,放电频率增加23.92±7.03%(P0.05),8-OH-DAPT则使这些神经元的基础放电频率明显降低(降低：19.28±3.45%,P0.05)。此外,向同一个丘脑底核神经元先后注射RO-600175和ML-10302,均使神经元放电频率明显升高(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电生理实验中,损毁侧记录到的38个丘脑底核神经元中,0.1mmM5-HT可使其中25个神经元的自发放电频率平均升高31.12±4.27%；另外13个丘脑底核神经元的兴奋性被5-HT抑制,放电频率平均降低23.10±7.05%。分别微量加压注射5-HT1B受体激动剂CP-93129、5-HT2C受体激动剂RO-600175和5-HT4受体激动剂ML-10302可使丘脑底核神经元放电频率升高28.76-4.43%、32.50±7.12%和26.16±6.61%。而注射5-HT1A受体激动剂8-OH-DPAT则使自发放电频率降低18.72±3.81%。 在帕金森病模型大鼠损毁对侧丘脑底核记录到的19个神经元中,0.1mM5-HT可使其中10个神经元的自发放电频率平均升高26.54±6.41%；另外9个丘脑底核神经元的放电频率降低21.94±5.95%。分别注射CP-93129、RO-600175和ML-10302可使丘脑底核神经元放电频率升高26.91±3.35%、32.75±8.72%和26.47-6.44%。而注射8-OH-DPAT则使放电频率降低23.27±5.17%。 3.与正常大鼠(放电频率：7.80±0.54Hz)相比,帕金森病模型大鼠损毁侧丘脑底核神经元的基础放电频率明显升高(放电频率：11.07±0.85Hz,P0.001)；并且放电模式总体分布也有改变(χ2=25.523,d.f.=2,P0.001),簇状放电明显增多(χ2=24.832,d.f=1,P0.001)而规则放电减少(χ2=8.917,d.f.=1,P0.01)。将帕金森病模型大鼠损毁侧丘脑底核神经元的基础放电频率及放电模式与损毁对侧(放电频率：7.83±0.63Hz)相比,也表现为损毁侧神经元放电频率明显升高(P0.01),且簇状放电增多(P0.01),规则放电减少(P0.01)。 为了进一步证实帕金森病状态下,大鼠丘脑底核神经元放电模式发生改变,我们对正常及帕金森病模型大鼠丘脑底核神经元自发放电ISI的CV和FF值进行分析,将帕金森病模型大鼠损毁侧神经元的CV值,分别与正常大鼠及未损毁侧神经元相比,损毁侧神经元的CV值明显升高,均有统计学意义(正常：0.68±0.05；损毁对侧：0.66±0.08；损毁侧：1.00±0.09,P0.01),同时也观察到FF值的升高,有显著差异(正常：0.21±0.04；损毁对侧：0.16±0.05；损毁侧：0.39±0.08,P0.05)。 同时,我们将5-HT及4种受体激动剂对帕金森病模型大鼠损毁侧丘脑底核神经元的电生理效应与正常大鼠相比较,结果显示均无显著差异(P0.05)。 4.在行为学实验中,大鼠腹腔注射氟哌啶醇能产生相对稳定的僵直症状,双侧丘脑底核微量注射0.1mM5-HT(0.3μl/侧)能够显著缩短氟哌啶醇导致的大鼠僵直时间,与注射生理盐水相比有显著统计学意义(P0.05)。 5.利用免疫组织化学技术,对正常大鼠丘脑底核神经元5-HT1B、5-HT2C、5-HT4和5-HT1A受体进行染色。在大鼠丘脑底核内均观察到5-HT1B、5-HT2C、5-HT4和5-HT1A4种受体亚型明显的阳性表达。 结论：电生理学研究结果显示外源性5-HT可以改变正常大鼠和帕金森病模型大鼠丘脑底核神经元的兴奋性,产生使放电频率升高和降低的双重效应。5-HT1B、5-HT2c和5-HT4受体被激活可以增加丘脑底核神经元放电频率,而5-HT1A受体的激活却对神经元放电频率起抑制作用。在帕金森病状态下,与正常大鼠相比,虽然丘脑底核神经元基础放电频率加快且簇状放电增多,但5-HT及相应受体激动剂对神经元的生物学效应却没有明显改变。形态学研究显示正常大鼠丘脑底核表达丰富的5-HT1B、5-HT2C、5-HT4和5-HT1A受体。 本实验结果为深入探讨丘脑底核5-HT的生物学效应及其在帕金森病的发生和防治中的作用提供了实验依据。
[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.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：Q424;R742.5
本文编号：2183975
[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.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：Q424;R742.5
【参考文献】
相关期刊论文 前1条
1 张巧俊;高蕊;刘健;刘娅萍;王爽;;帕金森病大鼠中缝背核5-羟色胺能神经元电活动的变化[J];生理学报;2007年02期
，本文编号：2183975
本文链接：https://www.wllwen.com/yixuelunwen/shenjingyixue/2183975.html
最近更新
教材专著
