阿立哌唑对中脑皮层多巴胺神经元电生理学特性及递质释放的影响

发布时间：2018-06-30 22:11

本文选题：阿立哌唑 + 中脑皮层　；参考：《山东大学》2013年硕士论文

【摘要】：精神分裂症是临床上发病率高、治愈率低的精神性顽疾,其发病机制与脑内多巴胺(dopamine, DA)、5-羟色胺和谷氨酸等多种物质异常有关,其中,DA递质失衡在精神分裂症中的作用为多数研究者认可。神经解剖学的研究发现,中脑多巴胺能神经元在发育过程中投射至不同的脑区,分化为功能不同的神经元亚群。其中,中脑皮层多巴胺神经元由腹侧被盖区(ventral tegmental area, VTA)特异性投射至前额叶皮质区,建立了中脑皮层多巴胺神经通路,该通路的异常是导致精神分裂症等精神疾患的解剖学基础。研究表明,中脑皮层DA神经元具有特定的电生理学特征,它们的自发放电频率大多数情况下要比中脑纹状体DA神经元快,而且在幅度不断增加的超极化电流注射的基础上,中脑皮层DA神经元显示出不明显的“sag”。中脑DA神经元可以从位于黑质和VTA的胞体或树突部位释放DA,激活其自身D2受体,可抑制细胞放电并降低DA释放。但至今对向脑前额叶皮质投射的中脑皮层DA神经元具体的电生理学特性及DA释放的调控仍不清楚。阿立哌唑是一类新型的抗精神病药物,可有效缓解精神分裂症病人症状,并具有神经保护功能。临床研究表明,阿立哌唑除兴奋5-HT1A受体、拮抗5-HT2A受体外,还可部分激动多巴胺D2受体。新近的文献报道,阿立哌唑对高K+引起的PC12细胞DA释放具有抑制作用。通过体内细胞记录发现,阿立哌唑可降低体内VTA内DA神经元的自发放电活性。激活G蛋白偶联的内向整流K+通道(G-protein-coupled inwardly rectifying potassium, GIRK),可抑制VTA和黑质区神经元的DA释放。目前,对于阿立哌唑是否通过影响中脑皮层DA神经元的DA释放及电兴奋性而发挥治疗精神分裂症的作用尚无报道。基于上述理念,我们推测阿立哌唑在治疗精神分裂症方面的机制,可能与其影响中脑皮层DA神经元电生理学特性及DA释放有关。为了证实这一设想,本实验进行了以下实验： 1、在立体定位仪引导下,应用神经逆行示踪技术将荧光示踪剂定点注射至鼠脑前前额叶皮质,该区域是中脑皮层DA神经元的投射靶区。通过轴浆逆行运输,可标记向靶区投射的中脑皮层DA神经元,并进行酪氨酸羟化酶(tyrosine hydroxylase, TH)免疫组织化学染色鉴定。 2、取标记鼠中脑组织,制备脑片,采用脑片膜片钳技术,在全细胞记录模式下,通过玻璃电极记录加入不同浓度阿立哌唑对中脑皮层DA神经元自发和诱发动作电位的幅度、频率、宽度的影响,并检测静息电位和阈值等方面的变化。 3、检测阿立哌唑对中脑皮层DA神经元外向K+电流变化的影响,分析阿立哌唑调节中脑皮层DA神经元兴奋性的可能机制。 4、在制备的中脑组织切片上,应用碳纤维电极技术检测阿立哌唑对标记的中脑皮层DA神经元DA释放的影响。在780mV恒压下,碳纤维电极可将DA氧化成多巴胺正醌,而DA氧化所产生的电流可被碳纤维电极检测到,从而将DA递质的变化转换成了伏安图谱,可达到纳摩尔级和亚秒级的精度；并应用DA标准液测试碳纤维电极情况。 5、进行在体实验研究,在脑VTA区注射阿立哌唑后,经碳纤维电极技术检测中脑皮层DA神经元投射靶区前额叶皮质内DA释放的变化情况。结果显示： 1、荧光示踪剂可准确标记向前额叶皮质区投射的中脑皮层DA神经元。绿色荧光示踪剂分布于神经元的胞浆内,亦可在神经元轴突内发现示踪剂颗粒。TH免疫组织化学染色显示,标记细胞呈TH阳性,这些DA神经元主要分布在VTA区。 2、应用脑片钳技术检测了不同浓度的阿立哌哗对标记中脑皮层DA神经元自发动作电位和诱发动作电位的影响,在灌流液中加入10μM的阿立哌唑后,可明显降低自发动作电位的频率(P0.05)和幅度(P0.01),与加药前比较,静息电位和阈值均明显降低(P0.05)；给予标记细胞300pA,500ms的电流刺激,可明显诱发出频率为30±1.15Hz,幅度为55.5±0.64mV的诱发动作电位,10μM的阿立哌唑明显降低其频率和幅度(P0.05),高浓度的阿立哌哗(100μM)可使自发及诱发动作电位消失。结果显示,阿立哌唑可抑制中脑皮层DA神经元的兴奋性。 3、为进一步分析阿立哌唑抑制中脑皮层DA神经元兴奋性的机制,我们又对外向K+电流做了检测,发现灌流液中加入10μM阿立哌唑可以刺激中脑皮层DA神经元的钾电流升高(P0.05),用灌流液洗去阿立哌唑后,钾电流恢复。 4、在脑片灌流液中加入阿立哌唑,经碳纤维电极可检测到标记DA神经元DA释放产生的氧化电流,与加药前比较,电流幅度(P0.05)和曲线下面积(P0.01)均明显减小。表明阿立哌唑可以抑制脑片中标记中脑皮层DA神经元的DA释放。实验中加以70mM高K+溶液作为阳性对照。当加在电极上的膜钳制电位跃迁至780mV电压时,碳纤维电极检测的氧化电流波谱上会有一上冲形成的尖峰；当钳制电位回到0mV时,尖峰消失。 5、在体实验中,向VTA区注射10μM阿立哌唑,应用碳纤维电极技术检测中脑皮层DA神经元的投射靶区前额叶皮质内DA释放所产生的氧化电流。加药2h后,检测到前额叶皮质区DA的氧化电流降低；3h时,氧化电流降至最低；直至8h时,电流恢复至加药前水平。综上所述,本实验结果表明阿立哌唑是通过兴奋外向K+电流来抑制中脑皮层DA神经元自发动作电位及诱发动作电位的产生,进而抑制中脑皮层DA神经元的兴奋性,降低中脑皮层DA神经元DA的分泌,从而发挥精神分裂症的治疗作用。
[Abstract]:Schizophrenia is a mental disease with high clinical incidence and low cure rate. Its pathogenesis is related to many substances such as dopamine (dopamine, DA), 5- serotonin and glutamic acid in the brain. Among them, the role of DA transmitter imbalance in schizophrenia is recognized by most researchers. Neuroanatomy studies found that dopamine in the middle brain It is projected into different brain regions during the development process and differentiating into different functional subgroups of neurons. The dopamine neurons in the middle cerebral cortex are specifically projected from the ventral tegmental area (ventral tegmental area, VTA) to the prefrontal cortex, and the dopamine translocation pathway in the middle cerebral cortex is established, and the abnormalities of this pathway lead to schizophrenia and so on. The anatomical basis of mental disorders shows that the DA neurons in the middle cerebral cortex have specific electrophysiological characteristics, and their spontaneous discharge frequencies are mostly faster than the DA neurons in the mesencephalic striatum, and the DA neurons in the middle cerebral cortex show an indistinct "sag" on the basis of an increasing amplitude of hyperpolarized current injection. The DA neurons in the mesencephalon can release DA from the cells or dendrites of the substantia nigra and VTA, activate their own D2 receptors, inhibit cell discharge and reduce DA release. However, the specific electrophysiological characteristics of the mesencephalic cortical DA neurons projecting to the cerebral prefrontal cortex and the regulation of DA release are still unclear.
Aripiprazole is a new type of antipsychotic drug, which can effectively alleviate the symptoms of schizophrenia and have neuroprotective functions. The clinical study shows that aripiprazole is an exexcitated 5-HT1A receptor, antagonistic 5-HT2A to the in vitro, and partly excitated the dopamine D2 receptor. Recently, aripiprazole has reported the release of DA from PC12 cells caused by high K+. In vivo cell recording, aripiprazole can reduce the spontaneous discharge activity of DA neurons in VTA in vivo. Activation of the G protein coupled inward rectifier K+ channel (G-protein-coupled inwardly rectifying potassium, GIRK) can inhibit DA release of VTA and the Shen Jing element in substantia nigra. Currently, it is possible for aripiprazole to pass through the passage of aripiprazole. The effects of DA release and electrical excitability on the DA neurons in the mesencephalon play a role in the treatment of schizophrenia.
Based on these ideas, we speculate that the mechanism of aripiprazole in the treatment of schizophrenia may be related to its effects on the electrophysiological characteristics of the mesencephalic DA neurons and the release of DA.
1, under the guidance of the stereotaxis, the fluorescent tracer was injected into the prefrontal cortex of the rat prefrontal cortex with the retrograde tracing technique, which was the target area of the DA neurons in the mesencephalon. Through retrograde axonal transport, the DA neurons of the mesencephalic cortex projecting into the target area were labeled, and the tyrosine hydroxylase (tyrosine hydroxylase, TH) was carried out. Identification of immuno histochemical staining.
2, the rat mesencephalic tissue was marked and brain slices were prepared, and the brain slice clamp technique was used to record the effects of aripiprazole on the amplitude, frequency and width of the evoked action potential of DA neurons in the middle cerebral cortex under the whole cell recording mode, and the changes in resting potential and threshold were detected.
3, to detect the effect of aripiprazole on the changes of outward K+ currents in the DA neurons of the mesencephalon, and to analyze the possible mechanism of aripiprazole regulating the excitability of DA neurons in the mesencephalon.
4, the effect of aripiprazole on the release of DA in the labeled mesencephalic DA neurons was detected by carbon fiber electrode technique on the prepared mesencephalic tissue section. Under the constant pressure of 780mV, the carbon fiber electrode can oxidize DA to dopamine quinone, and the current produced by DA oxidation can be detected by the carbon fiber electrode, thus converting the change of DA transmitter into a change. The voltammetric map can reach the accuracy of nanomolar and sub second level, and the DA standard solution is used to test the carbon fiber electrode.
5, the changes of DA release in the prefrontal cortex of the DA neurons in the middle cerebral cortex were detected by the carbon fiber electrode technique after the injection of aripiprazole in the brain VTA area.
The results show that:
1, the fluorescent tracer can accurately mark the mesencephalic DA neurons projecting in the frontal cortex. The green fluorescent tracer is distributed in the cytoplasm of the neurons, and the tracer particles.TH immuno histochemical staining can be found in the axon of the neuron, and the labeled cells are TH positive, and these DA neurons are mainly distributed in the VTA region.
2, the effects of different concentrations of aripiperm on the spontaneous action potential and induced action potential of the DA neurons in the middle cerebral cortex were detected by using the technique of brain slice forceps. The frequency of spontaneous action potential (P0.05) and amplitude (P0.01) were obviously reduced after adding 10 mu of aripiprazole in the perfusion fluid. The resting potential and threshold value were all clearly compared with those before the addition of drugs. Significantly decreased (P0.05); the current stimulation of labeled cells 300pA, 500ms could obviously induce the induced action potential of 30 + 1.15Hz, and the amplitude was 55.5 + 0.64mV. The frequency and amplitude of aripiprazole at 10 u M decreased significantly (P0.05). The high concentration of aripipric (100 mu M) could make spontaneous and induced action potential disappear. Azole can inhibit the excitability of DA neurons in the middle cerebral cortex.
3, in order to further analyze the mechanism of aripiprazole to inhibit the excitatory of DA neurons in the middle cerebral cortex, we also detected the K+ current. It was found that the addition of 10 u M alpiprazole in the perfusion fluid could stimulate the potassium current of the DA neurons in the mesencephalon (P0.05). The potassium current was restored after alidazole was washed with perfusion fluid.
4, aripiprazole was added into the cerebral perfusion fluid, and the oxidation current produced by the release of the labeled DA neuron DA was detected by the carbon fiber electrode. The current amplitude (P0.05) and the area under the curve (P0.01) decreased significantly compared with that before the addition of the drug, indicating that aripiprazole could inhibit the DA release of the DA neurons in the brain slices. In the experiment, 70mM The high K+ solution is used as a positive control. When the membrane potential of the electrode is transferred to the 780mV voltage, the peak of the oxidation current in the carbon fiber electrode will have a sharp peak, and the peak will disappear when the clamp potential is back to 0mV.
5, in the experiment, 10 M aripiprazole was injected into the VTA area, and the carbon fiber electrode technique was used to detect the oxidation current produced by the release of DA in the prefrontal cortex of the DA neurons of the middle cerebral cortex. After adding 2h, the oxidation current of DA in the prefrontal cortex was detected and the oxidation current decreased to the lowest when 3h, and the current was restored until 8h. Level before adding medicine.
To sum up, the results of this experiment show that aripiprazole inhibits the spontaneous action potential and induced action potential of DA neurons in the middle cerebral cortex through extroverted K+ current, and then inhibits the excitability of DA neurons in the middle cerebral cortex and reduces the secretion of DA in the DA neurons of the middle cerebral cortex, thus giving play to the therapeutic effect of schizophrenia.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.3

【参考文献】