本文選題：可卡因　切入點(diǎn)：行為敏化　出處：《華中科技大學(xué)》2015年博士論文

【摘要】：第一部分伏隔核區(qū)AMPA受體轉(zhuǎn)運(yùn)對(duì)可卡因所致大鼠行為敏化的影響及機(jī)制 目的：大量的研究證明可卡因引起的行為敏化與谷氨酸傳遞高度相關(guān),反復(fù)給予可卡因再戒斷增加伏隔核(nucleus accumbens, NAc)區(qū)突觸膜上的α-氨基-3-羥基-5-甲基-4-異VA唑丙酸受體(a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, AMPARs)水平,但AMPARs與行為敏化之間的關(guān)系仍不清楚。N-乙烷馬來(lái)酰亞胺敏感因子(N-ethylmaleimide-sensitive factor, NSF)與α-氨基-3-羥基-5-甲基-4-異VA唑丙酸受體亞單位2(AMPA receptor subunit2, GluA2)間的相互作用被認(rèn)為在維持AMPARs受體的突觸傳遞中非常重要,但NSF與GluA2間的相互作用在可卡因所致大鼠行為敏化中的研究鮮有報(bào)道。本研究旨在探討NSF與GluA2的相互作用與可卡因行為敏化的關(guān)系,進(jìn)一步闡明可卡因行為敏化的病理機(jī)制。 方法：采用自發(fā)活動(dòng)實(shí)驗(yàn)評(píng)價(jià)大鼠的行為敏化、BS3交聯(lián)檢測(cè)AMPA受體的膜表達(dá)、免疫共沉淀檢測(cè)NSF與GluA2之間的結(jié)合力、免疫熒光檢測(cè)NSF與GluA2之間的共定位。 結(jié)果：給予大鼠慢性腹腔注射可卡因建立行為敏化動(dòng)物模型。免疫熒光實(shí)驗(yàn)發(fā)現(xiàn)GluA2和NSF蛋白的共定位增加,免疫共沉淀實(shí)驗(yàn)發(fā)現(xiàn)GluA2和NSF的結(jié)合增加(sal:100.00±16.82; coc:155.38±19.74; n=4; p0.05vssal)。在可卡因強(qiáng)化之前伏隔核區(qū)注射干擾GluA2和NSF結(jié)合的多肽TAT-pep-R845A可增強(qiáng)大鼠對(duì)可卡因的敏化行為(aCSF+coc:23526.25±2002.80; TAT-pep+coc:22567.27±1900.80; TAT-pep-R845A+coc:30283.93±987.41; n=6-12rats; p0.01vs other experimental groups)。BS3交聯(lián)法發(fā)現(xiàn)TAT-pep-R845A孵育行為敏化大鼠腦片可抑制GluA2的膜表達(dá)(TAT-pep:100.00±6.04:TAT:pep-R845A:54.80±6.80；n.5；p0.05vs control peptide).伏隔核區(qū)定位注射TAT-pep-R845A亦可抑制GluA2的膜表達(dá)(TAT-pep:100.00±9.40；TAT-pep-R845A:68.75±12.10； n=5rats:p0.05vs control peptide). 結(jié)論：GluA2和NSF結(jié)合增強(qiáng)促進(jìn)GluA2的膜表達(dá)是機(jī)體對(duì)可卡因的一種抵抗,干預(yù)兩者間的結(jié)合可增強(qiáng)大鼠對(duì)可卡因的敏化行為,其機(jī)制可能是通過(guò)抑制GluA2的膜表達(dá)。 第二部分背側(cè)紋狀體區(qū)AMPAK受體降解對(duì)可卡因所致大鼠攝藥行為的影響及機(jī)制 目的：藥物成癮是長(zhǎng)期慢性使用成癮性藥物而引起的強(qiáng)迫性覓藥行為。背側(cè)紋狀體是調(diào)節(jié)強(qiáng)迫性攝藥行為的主要腦區(qū)。以往的研究多關(guān)注NAc區(qū)的谷氨酸投射與可卡因覓藥行為的關(guān)系。而習(xí)慣性的攝藥行為與背側(cè)紋狀體區(qū)的動(dòng)機(jī)環(huán)路更加密切。因此本研究探討背側(cè)紋狀體區(qū)域AMPAR在可卡因強(qiáng)迫性攝藥行為中的作用及機(jī)制。 方法：可卡因自身給藥檢測(cè)動(dòng)物的攝藥行為；實(shí)時(shí)定量pCR檢測(cè)GluAl,2,3的mRNA水平；免疫印跡檢測(cè)相關(guān)蛋白的表達(dá)；腦區(qū)注射病毒或者藥物；免疫熒光檢測(cè)相關(guān)病毒表達(dá)情況和蛋白表達(dá)情況。 結(jié)果：可卡因自身給藥訓(xùn)練后大鼠背側(cè)紋狀體區(qū)域GluAl的表達(dá)出現(xiàn)下調(diào)(saline:100.00±5.69%；cocaine:60.00±2.07%;n=8；p0.01vs saline).而過(guò)表達(dá)GluAl增加大鼠對(duì)可卡因的攝入(n=8-9rats；p0.05,p0.01vs the sameday in lenti-control rats).同時(shí)研究發(fā)現(xiàn),可卡因自身給藥訓(xùn)練增加大鼠背側(cè)紋狀體區(qū)域自噬標(biāo)志物L(fēng)C3II蛋白表達(dá)(saline:100.00±17.17%;cocaine limited:169.44±25.17%；cocaine prolonged:165.85±20.28%；n=4-10rats；p0.05,p0.01vs saline),降低自噬底物蛋白62(protein62,p62)的表達(dá)(saline:100.00±5.35%；cocaine:35.23±3.30%;n=4-10rats p0.05,p0.01vs saline)。 結(jié)論：可卡因自身給藥引起的大鼠背側(cè)紋狀體區(qū)域GluAl的表達(dá)下調(diào)。
[Abstract]:The effect and mechanism of AMPA receptor transport in the nucleus accumbens on cocaine induced behavioral sensitization in the first part of the nucleus accumbens
Objective: it has been proven that cocaine induced behavioral sensitization and glutamate transmission are highly correlated, repeated cocaine withdrawal and an increase in the nucleus accumbens (nucleus accumbens, NAc) on the membrane of synaptic alpha amino -3- hydroxy -5- methyl -4- ISO VA acid receptor (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, AMPARs) level, but the relationship between AMPARs and behavioral sensitization remains unclear.N- ethane maleimide sensitive factor (N-ethylmaleimide-sensitive factor, NSF -3-) and alpha amino hydroxy -5- methyl -4- ISO VA acid receptor subunit 2 (AMPA receptor, subunit2, GluA2) interactions are considered in the maintenance of AMPARs receptors in synaptic transmission is very important, but the interaction of NSF between GluA2 and little research in behavior of cocaine induced sensitization in the rat in the report. This study aims to investigate the interaction between NSF and GluA2. The relationship with cocaine behavioral sensitization further clarifies the pathological mechanism of cocaine behavioral sensitization.
Methods: spontaneous activity test was used to evaluate behavioral sensitization in rats. BS3 cross-linking was used to detect the expression of AMPA receptor, and the binding force between NSF and GluA2 was detected by immunoprecipitation. The co localization between NSF and GluA2 was detected by immunofluorescence.
Results: the rats were given intraperitoneal injection to establish chronic cocaine sensitization animal model. Immunofluorescence test showed that GluA2 and NSF protein co localization increases, it is found that increasing the combination of GluA2 and NSF co immunoprecipitation (sal:100.00 + 16.82 + 19.74; coc:155.38; n=4; p0.05vssal). In cocaine reinforcement before V polypeptide TAT-pep-R845A septum injection region the combination of NSF and GluA2 interference can enhance sensitized rat behavior for cocaine (aCSF+coc:23526.25 + TAT-pep+coc:22567.27 + 2002.80; 1900.80 + 987.41; TAT-pep-R845A+coc:30283.93; n= 6-12rats; p0.01vs other experimental groups.BS3) cross-linking method that the expression of TAT-pep-R845A were incubated with behavioral sensitization in rat brain slices inhibited GluA2 membrane (TAT-pep:100.00 + 6.04:TAT:pep-R845A:54.80 + 6.80; N.5; p0.05vs control peptide). The nucleus accumbens injection of TAT-pep-R845A also can inhibit the GluA2 film Expression (TAT-pep:100.00 + 9.40; TAT-pep-R845A:68.75 + 12.10; n=5rats:p0.05vs control peptide).
Conclusion: the combination of GluA2 and NSF enhances the expression of GluA2, which is a resistance of the body to cocaine. Intervening the combination between them can enhance the sensitization behavior of cocaine in rats. The mechanism may be by inhibiting the expression of GluA2 membrane.
The effect and mechanism of AMPAK receptor degradation in the second part of the dorsal striatum on the behavior of rats induced by cocaine
Objective: drug addiction is a chronic compulsive use of addictive drugs caused by drug seeking behavior. The dorsal striatum is regulated compulsive main brain areas taken delivery behavior. Previous studies pay more attention to the relationship between glutamate projection and NAc region of the cocaine drug seeking behavior. The behavior and drug uptake in dorsal striatum area of habitual the motivation of the loop more closely. This study of dorsal striatum region AMPAR effect and mechanism of forced drug behavior in cocaine intake.
Methods: cocaine self medication was used to detect the drug taking behavior of animals, real-time quantitative pCR was used to detect the mRNA level of GluAl and 2,3, the expression of related proteins was detected by Western blotting, virus or drugs were injected into the brain area, and the expression and expression of virus related virus were detected by immunofluorescence.
Results: the expression of cocaine to the dorsal striatum of rats GluAl drug after training down (saline:100.00 + 5.69% + 2.07%; cocaine:60.00; n=8; p0.01vs saline). And the expression of GluAl increased intake of cocaine in rats (n=8-9rats; P0.05, p0.01vs the sameday in lenti-control rats). At the same time, the study found that cocaine itself administration training increased in rat dorsal striatum region of autophagy marker LC3II expression protein (saline:100.00 + 17.17% cocaine + 25.17% cocaine; limited:169.44; prolonged:165.85 + 20.28%; n=4-10rats; P0.05, p0.01vs, saline) reduced autophagy substrate protein 62 (protein62, p62) expression (saline:100.00 + 5.35% cocaine:35.23 + 3.30% n=4-10rats; P0.05, p0.01vs; saline).
Conclusion: the expression of GluAl in the dorsal striatum region of rats induced by cocaine administration is down regulated.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R965

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