本文選題：阿立哌唑 + 中腦皮層��； 參考：《山東大學》2013年碩士論文

【摘要】：精神分裂癥是臨床上發(fā)病率高、治愈率低的精神性頑疾,其發(fā)病機制與腦內(nèi)多巴胺(dopamine, DA)、5-羥色胺和谷氨酸等多種物質異常有關,其中,DA遞質失衡在精神分裂癥中的作用為多數(shù)研究者認可。神經(jīng)解剖學的研究發(fā)現(xiàn),中腦多巴胺能神經(jīng)元在發(fā)育過程中投射至不同的腦區(qū),分化為功能不同的神經(jīng)元亞群。其中,中腦皮層多巴胺神經(jīng)元由腹側被蓋區(qū)(ventral tegmental area, VTA)特異性投射至前額葉皮質區(qū),建立了中腦皮層多巴胺神經(jīng)通路,該通路的異常是導致精神分裂癥等精神疾患的解剖學基礎。研究表明,中腦皮層DA神經(jīng)元具有特定的電生理學特征,它們的自發(fā)放電頻率大多數(shù)情況下要比中腦紋狀體DA神經(jīng)元快,而且在幅度不斷增加的超極化電流注射的基礎上,中腦皮層DA神經(jīng)元顯示出不明顯的“sag”。中腦DA神經(jīng)元可以從位于黑質和VTA的胞體或樹突部位釋放DA,激活其自身D2受體,可抑制細胞放電并降低DA釋放。但至今對向腦前額葉皮質投射的中腦皮層DA神經(jīng)元具體的電生理學特性及DA釋放的調(diào)控仍不清楚。 阿立哌唑是一類新型的抗精神病藥物,可有效緩解精神分裂癥病人癥狀,并具有神經(jīng)保護功能。臨床研究表明,阿立哌唑除興奮5-HT1A受體、拮抗5-HT2A受體外,還可部分激動多巴胺D2受體。新近的文獻報道,阿立哌唑對高K+引起的PC12細胞DA釋放具有抑制作用。通過體內(nèi)細胞記錄發(fā)現(xiàn),阿立哌唑可降低體內(nèi)VTA內(nèi)DA神經(jīng)元的自發(fā)放電活性。激活G蛋白偶聯(lián)的內(nèi)向整流K+通道(G-protein-coupled inwardly rectifying potassium, GIRK),可抑制VTA和黑質區(qū)神經(jīng)元的DA釋放。目前,對于阿立哌唑是否通過影響中腦皮層DA神經(jīng)元的DA釋放及電興奮性而發(fā)揮治療精神分裂癥的作用尚無報道。 基于上述理念,我們推測阿立哌唑在治療精神分裂癥方面的機制,可能與其影響中腦皮層DA神經(jīng)元電生理學特性及DA釋放有關。為了證實這一設想,本實驗進行了以下實驗： 1、在立體定位儀引導下,應用神經(jīng)逆行示蹤技術將熒光示蹤劑定點注射至鼠腦前前額葉皮質,該區(qū)域是中腦皮層DA神經(jīng)元的投射靶區(qū)。通過軸漿逆行運輸,可標記向靶區(qū)投射的中腦皮層DA神經(jīng)元,并進行酪氨酸羥化酶(tyrosine hydroxylase, TH)免疫組織化學染色鑒定。 2、取標記鼠中腦組織,制備腦片,采用腦片膜片鉗技術,在全細胞記錄模式下,通過玻璃電極記錄加入不同濃度阿立哌唑對中腦皮層DA神經(jīng)元自發(fā)和誘發(fā)動作電位的幅度、頻率、寬度的影響,并檢測靜息電位和閾值等方面的變化。 3、檢測阿立哌唑對中腦皮層DA神經(jīng)元外向K+電流變化的影響,分析阿立哌唑調(diào)節(jié)中腦皮層DA神經(jīng)元興奮性的可能機制。 4、在制備的中腦組織切片上,應用碳纖維電極技術檢測阿立哌唑對標記的中腦皮層DA神經(jīng)元DA釋放的影響。在780mV恒壓下,碳纖維電極可將DA氧化成多巴胺正醌,而DA氧化所產(chǎn)生的電流可被碳纖維電極檢測到,從而將DA遞質的變化轉換成了伏安圖譜,可達到納摩爾級和亞秒級的精度；并應用DA標準液測試碳纖維電極情況。 5、進行在體實驗研究,在腦VTA區(qū)注射阿立哌唑后,經(jīng)碳纖維電極技術檢測中腦皮層DA神經(jīng)元投射靶區(qū)前額葉皮質內(nèi)DA釋放的變化情況。 結果顯示： 1、熒光示蹤劑可準確標記向前額葉皮質區(qū)投射的中腦皮層DA神經(jīng)元。綠色熒光示蹤劑分布于神經(jīng)元的胞漿內(nèi),亦可在神經(jīng)元軸突內(nèi)發(fā)現(xiàn)示蹤劑顆粒。TH免疫組織化學染色顯示,標記細胞呈TH陽性,這些DA神經(jīng)元主要分布在VTA區(qū)。 2、應用腦片鉗技術檢測了不同濃度的阿立哌嘩對標記中腦皮層DA神經(jīng)元自發(fā)動作電位和誘發(fā)動作電位的影響,在灌流液中加入10μM的阿立哌唑后,可明顯降低自發(fā)動作電位的頻率(P0.05)和幅度(P0.01),與加藥前比較,靜息電位和閾值均明顯降低(P0.05)；給予標記細胞300pA,500ms的電流刺激,可明顯誘發(fā)出頻率為30±1.15Hz,幅度為55.5±0.64mV的誘發(fā)動作電位,10μM的阿立哌唑明顯降低其頻率和幅度(P0.05),高濃度的阿立哌嘩(100μM)可使自發(fā)及誘發(fā)動作電位消失。結果顯示,阿立哌唑可抑制中腦皮層DA神經(jīng)元的興奮性。 3、為進一步分析阿立哌唑抑制中腦皮層DA神經(jīng)元興奮性的機制,我們又對外向K+電流做了檢測,發(fā)現(xiàn)灌流液中加入10μM阿立哌唑可以刺激中腦皮層DA神經(jīng)元的鉀電流升高(P0.05),用灌流液洗去阿立哌唑后,鉀電流恢復。 4、在腦片灌流液中加入阿立哌唑,經(jīng)碳纖維電極可檢測到標記DA神經(jīng)元DA釋放產(chǎn)生的氧化電流,與加藥前比較,電流幅度(P0.05)和曲線下面積(P0.01)均明顯減小。表明阿立哌唑可以抑制腦片中標記中腦皮層DA神經(jīng)元的DA釋放。實驗中加以70mM高K+溶液作為陽性對照。當加在電極上的膜鉗制電位躍遷至780mV電壓時,碳纖維電極檢測的氧化電流波譜上會有一上沖形成的尖峰；當鉗制電位回到0mV時,尖峰消失。 5、在體實驗中,向VTA區(qū)注射10μM阿立哌唑,應用碳纖維電極技術檢測中腦皮層DA神經(jīng)元的投射靶區(qū)前額葉皮質內(nèi)DA釋放所產(chǎn)生的氧化電流。加藥2h后,檢測到前額葉皮質區(qū)DA的氧化電流降低；3h時,氧化電流降至最低；直至8h時,電流恢復至加藥前水平。 綜上所述,本實驗結果表明阿立哌唑是通過興奮外向K+電流來抑制中腦皮層DA神經(jīng)元自發(fā)動作電位及誘發(fā)動作電位的產(chǎn)生,進而抑制中腦皮層DA神經(jīng)元的興奮性,降低中腦皮層DA神經(jīng)元DA的分泌,從而發(fā)揮精神分裂癥的治療作用。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R749.3

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相關期刊論文 前1條

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本文編號：2087012