【摘要】：第一部分氣體信號分子NO對可卡因戒斷所致的大鼠成癮記憶的影響及機制 目的：一氧化氮(NO)作為一種內源性氣體信號分子,在中樞神經系統(tǒng)發(fā)揮廣泛的生物學效應。有研究報道NO參與可卡因等藥物成癮的形成過程。然而,NO是否也在可卡因成癮的戒斷行為中發(fā)揮重要作用則尚未見報道。近年來的研究發(fā)現(xiàn),可卡因成癮戒斷會導致伏隔核(NAc)區(qū)突觸表面的a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid受體(AMPA受體)受體表達上調,而這種上調被認為與可卡因的行為敏化的維持有關。NO可通過其亞硝基化作用調節(jié)突觸部位AMPA受體的膜表達。因此,本實驗研究NO在可卡因成癮戒斷所致大鼠成癮記憶中的作用及其機制。 方法：采用蛋白交聯(lián)的western blotting技術研究大鼠NAc區(qū)AMPA受體亞基(GluR1和GluR2)在突觸部位的膜表達情況；采用腦片膜片鉗技術和場電位記錄方法研究NAc區(qū)AMPA受體電流和LTD的變化情況；采用自發(fā)活動及糖水偏好實驗觀察可卡因成癮戒斷所致的大鼠行為學變化。 結果：可卡因戒斷導致大鼠NAc區(qū)突觸部位GluR1和GluR2亞基的蛋白表達出現(xiàn)不成比例的上調,表現(xiàn)為突觸表面GluR2的表達相對較少、鈣通透性AMPA受體介導的電流增加。進一步研究發(fā)現(xiàn),戒斷后NAc區(qū)nNOS的表達、NO的含量,以及N-ethylmaleimide-sensitive factor (NSF)的亞硝基化水平均有所增加,提示可卡因戒斷所致的NAc突觸表面GluR2亞基表達相對較少可能是因NSF功能不足所致。外源性使用NO供體可增加戒斷大鼠NAc區(qū)的NSF亞硝基化水平并選擇性上調突觸部位GluR2的含量,從而恢復GluR1和GluR2膜表達平衡及鈣通透性AMPA受體電流。特異性干擾NSF-GluR2結合的生物肽可阻斷NO的這種效應,提示NO的作用依賴于NSF與GluR2亞基的結合。場電位實驗也證實NO是通過促進NSF亞硝基化并上調GluR2的膜表達,進而逆轉戒斷后NAc區(qū)的突觸可塑性變化(長時程抑制,LTD)。另外,外源性給予NO供體和亞硝酸鈉均可通過增加NAc區(qū)NSF與GluR2的結合能力來阻斷戒斷后大鼠的行為敏化。特異性阻斷NAc區(qū)GluR2亞基內化過程也可減輕戒斷大鼠的敏化行為,提示突觸表面GluR2表達相對不足是行為敏化的重要原因。 結論：可卡因戒斷引起的NAc區(qū)NSF亞硝基化水平和突觸部位GluR2亞基表達增加是大腦出現(xiàn)的一種內源性代償機制,從而限制機體對可卡因的長時程異常行為反應。靶向性調控NAc區(qū)NO的生成及NSF的硝基化水平可能是可卡因成癮治療的新策略。 第二部分氣體信號分子H2S對大鼠海馬區(qū)介導的學習記憶的影響及機制 目的：硫化氫(H2S)是一種內源性氣體信號分子,在生物體內發(fā)揮著廣泛的生物學效應。中樞神經系統(tǒng)的H2S作為一個突觸調節(jié)分子,具有神經保護作用。近來有研究證實,神經退行性疾病(如阿爾茨海默病、帕金森病等)患者或動物大腦H2S含量出現(xiàn)異常。H2S可易化突觸傳遞的長時程增強(LTP),但目前對于H2S與學習記憶的關系則尚未有相關的報道。本實驗旨在研究氣體信號分子H2S在大鼠海馬介導的記憶及突觸可塑性形成的作用及機制。 方法：采用亞甲基藍法檢測大鼠海馬組織H2S含量；條件性恐懼記憶和新事物認知實驗觀察大鼠的認知功能；腦片膜片鉗和場電位技術記錄大鼠海馬區(qū)NMDA受體介導的電流及LTP; Western blotting實驗研究大鼠海馬與突觸可塑性密切相關蛋白的表達水平。 結果：條件性驚恐學習訓練可明顯增加大鼠海馬區(qū)H2S的含量。H2S合成酶抑制劑可阻斷訓練引起的海馬H2S含量增加,并損傷海馬依賴的情景型恐懼記憶。外源性補充H2S可劑量依賴性的增強大鼠情景型恐懼記憶。同樣,抑制內源性H2S合成可損傷大鼠海馬依賴的新物體認知功能,而外源性H2S可促進動物的這種認知能力。電生理實驗發(fā)現(xiàn),H2S選擇性增強海馬區(qū)含NR2A亞基的NMDA受體介導的電流和NMDA受體依賴的海馬LTP；且NR2A特異性阻斷劑可取消H2S對LTP和認知功能的增強作用。Western blotting結果表明H2S的促認知作用與PKA、PKC、CaMKII和CREB等信號通路激活有關。 結論：作為一種內源性氣體信號分子,H2S在海馬依賴記憶和突觸可塑性形成過程中扮演著重要角色。該結果進一步加深了我們對H2S生理功能的理解,同時也提示H2S釋放藥物可能是認知障礙治療的一種新策略。 第三部分氣體信號分子H2S對大鼠杏仁核區(qū)介導的情感記憶的影響及機制 目的：杏仁核是產生、識別和調節(jié)情緒的關鍵腦區(qū),控制情感性學習及記憶行為。有研究發(fā)現(xiàn),額顳葉退變性疾病和阿爾茨海默病患者可出現(xiàn)杏仁核依賴的情感記憶異常；孤獨癥患兒的大腦杏仁核也存在功能障礙。因此,研究內源性物質在杏仁核中的功能具有重要意義。H2S作為一種內源性氣體信號分子,在海馬區(qū)扮演著神經調質的角色。但其是否也在杏仁核介導的情感記憶行為和突觸可塑性中發(fā)揮重要作用則尚未見相關報道。 方法：采用亞甲基藍法檢測大鼠腦組織H2S的含量；條件性恐懼記憶和條件性味覺厭惡實驗觀察H2S對大鼠情感記憶行為的影響；腦片膜片鉗和場電位技術記錄大鼠杏仁核區(qū)NMDA受體介導的電流及LTP; Western blotting和Golgi染色技術研究大鼠杏仁核區(qū)突觸結構情況。 結果：線索型恐懼學習訓練增加大鼠腦組織的H2S含量。外源性給予H2S供體可明顯增強杏仁核介導的線索型恐懼記憶,且這種效應在訓練后3周仍持續(xù)存在。此外,H2S可減緩恐懼記憶的消散,促進恐懼記憶自發(fā)性的恢復和復發(fā)。外源性給予H2S也可明顯增強杏仁核依賴的條件性味覺厭惡記憶。免疫組化實驗發(fā)現(xiàn)H2S合成酶胱硫醚-β-合成酶(CBS)在杏仁核區(qū)有大量表達,杏仁核內微量注射CBS抑制劑可損傷大鼠的線索型恐懼記憶。進一步實驗發(fā)現(xiàn),H2S可增加杏仁核區(qū)神經元的樹突棘密度,增加CREB活性和BDNF含量,并促進NR2B亞基的膜表達。電生理實驗發(fā)現(xiàn),H2S可選擇性增強丘腦-杏仁核通路含NR2B亞基的NMDA受體所介導的電流及NMDA受體依賴性LTP. NR2B特異性阻斷劑可取消H2S對LTP和認知功能的促進作用。結論：H2S通過增強含NR2B亞基的NMDA受體功能,從而起到增強情感記憶的作用。該研究為深入理解杏仁核及H2S的生理功能提供了實驗依據,也為將H2S釋放藥物開發(fā)成為治療情感障礙性疾病的藥物提供新思路。
[Abstract]:The effect and mechanism of NO in the first part of gas signal on the addiction and memory of rats induced by cocaine withdrawal Objective: Nitric oxide (NO), as an endogenous gas signal molecule, has a wide range of biological effects in the central nervous system It should be. It is reported that NO is involved in the formation of drug addiction, such as cocaine. However, whether NO also plays an important role in the withdrawal of cocaine addiction has not yet been reported In recent years, it has been found that the dependence of cocaine addiction leads to an up-regulation of the expression of a-amino-3-hydroxyxy-5-methyl-4-isooxazoleponate acid receptor (AMPA receptor) on the synaptic surface in the nucleus accumbens (NAc) region, and this up-regulation is considered to be associated with the maintenance of the behavioral sensitization of cocaine. OFF. NO can regulate the membrane form of the AMPA receptor at the synapse site by its nitroso-chemical action. Da. Therefore, this experiment study the role of NO in the addiction and memory of rats induced by cocaine addiction and its machine Methods: The expression of AMPA receptor subunit (GluR1 and GluR2) in the synapse of the rat NAc region was studied by using the western blotting technique of protein cross-linking, and the changes of AMPA receptor current and LTD in the NAc region were studied by the technique of the patch clamp and the field potential recording method. The behavior of the rats caused by the withdrawal of cocaine addiction was observed by spontaneous activity and sugar water preference experiment. Results: Cocaine withdrawal resulted in a disproportionate increase in the expression of GluR1 and GluR2 subunits in the synapses of the rat NAc region, showing a relatively low expression of GluR2 on the synaptic surface and an AMPA receptor mediated by calcium permeability. The results showed that the expression of nNOS in the NAc region after withdrawal, the content of NO, and the nitroso level of N-ethylmaleiide-sensitive factor (NSF) increased, suggesting that the expression of GluR2 subunit in the NAc synaptic surface due to the withdrawal of cocaine is relatively less likely to be due to the NSF work. Can increase the NSF nitroso level in the NAc region of the withdrawal rat and selectively increase the content of the GluR2 in the synapse site, thereby restoring the expression balance of the GluR1 and GluR2 membranes and the calcium permeability AMP. A receptor current. A biological peptide that specifically interferes with the binding of NSF-GluR2 can block this effect of NO, suggesting that the effect of NO is dependent on the NSF and GluR2 The binding of the subunits. The field potential experiment also confirmed that NO was by promoting NSF nitroso and up-regulating the membrane expression of GluR2, and then reversing the synaptic plasticity in the NAc region after withdrawal (long-time history inhibition). In addition, exogenous NO donor and sodium nitrite can block the rats after withdrawal by increasing the binding ability of NSF and GluR2 in the NAc region. The specific blocking of the internalization of GluR2 subunit in the NAc region can also reduce the sensitization behavior of the withdrawal rats, and suggest that the expression of GluR2 in the synaptic surface is relatively deficient. Conclusion: The NSF nitroso level in the NSF region and the increase of GluR2 subunit expression in the synapse of the NAc region caused by the withdrawal of cocaine are an endogenous compensatory mechanism in the brain, thus limiting the long-term effect of the body on the cocaine. The generation of NO in the NAc region and the nitrosation level of NSF may be cocaine. A new strategy for the treatment of addiction. The second part of the gas signal, H2S, is mediated by the rat hippocampus. The effect of learning and memory and the purpose of the mechanism: hydrogen sulfide (H2S) is an endogenous gas signal molecule, in the organism A wide range of biological effects. The central nervous system's H2S is a synaptic regulation. Molecular, neuroprotective. Recent studies have shown that neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, etc.) patients or animals The content of H2S in the brain is abnormal. The long-term potentiation (LTP) of the synaptic transmission is facilitated by the H2S, but at present, for H2S and learning and memory, The purpose of this experiment is to study the memory and synapses mediated by the gas signal molecules in the hippocampus of the rat. Effect and mechanism of plasticity formation. Methods: The content of H2S in the hippocampus of rats was detected by methylene blue method. The cognitive function of the rats was observed by the conditional fear memory and the new thing cognitive experiment. The brain-patch clamp and the field-potential technique were used to record the NM in the hippocampus of the rat. DA receptor-mediated current and LTP; Western blotting was used to study the rat hippocampal and synapses. The results showed that the conditional panic-learning exercise could the content of h2s in the hippocampus of the rat is obviously increased. the h2s synthetase inhibitor can block the increase of the h2s content in the hippocampus, And damage the hippocampal-dependent, situational-type, fear-memory. The exogenous supplemental H2S can be used in a dose-dependent manner. In the same way, the inhibition of endogenous H2S synthesis can damage the cognitive function of the new object in the hippocampus of the rat, while the exogenous H2S synthesis can damage the cognitive function of the new object that can damage the hippocampus of the rat. H2S can promote the cognitive ability of the animals. The electrophysiological experiments have found that the selective enhancement of H2S in the hippocampus is mediated by the NMDA receptor of the NR2A subunit and the NMDA receptor-dependent hippocampal LTP; and the NR2A-specific blocking agent can eliminate H2. The effect of S on LTP and cognitive function. Western blotting results show that the pro-cognitive function of H2S is similar to that of PKA, PKC and CaMKI. Conclusion: As an endogenous gas signal molecule, H2S is dependent on the memory of the hippocampus and The process of synaptic plasticity plays an important role. The result further deepens our understanding of the physiological function of H2S, and also prompts the release of H2S. The drug may be a new strategy for the treatment of cognitive disorders. The third part of the gas signal molecules, H2S, The effect and mechanism of the emotional memory mediated by amygdala in rats: the amygdala is the production, identification and modulation. The key brain regions of the mood are controlled by emotional learning and memory behavior. It has been found that the emotional memory of amygdala dependence can occur in the patients with degenerative disease and Alzheimer's disease. Abnormality; the brain amygdala in a child with autism also has a dysfunction. So, It is of great significance to study the function of endogenous substances in the amygdala. Gas signal molecules play a role in neuromodulation in the hippocampus, but are they also involved in the amygdala-mediated emotional memory behavior Methods: The content of H2S in the brain of rats was detected by the methylene blue method. The effect of H2S on the behavior of the rat's emotional memory was observed by the conditional fear memory and the conditioned taste aversion experiment. To record the NMDA receptor-mediated current and LTP in the amygdala of the rat; The synaptic structure of amygdala in rats was studied by Golgi staining. Results: The content of H2S in the brain of rats was increased by lead-type fear learning and training. in addition, that H2S may To slow the dissipation of the memory of fear and to promote the spontaneous recovery and recurrence of the memory of fear. The conditional taste aversion memory of the amygdala can also be obviously enhanced to the H2S. The expression of the S _ 2S-1-1-synthetase (CBS) in the amygdala is found by the immunohistochemical study, and the almonds The microinjection of CBS inhibitor in the nucleus can damage the lead-type fear memory of the rat. It is further found that H2S can increase the dendritic spine density of the neurons in the amygdala and increase the CR. The activity of EB and the content of BDNF and the expression of NR2B subunits were also promoted. The results showed that the selective enhancement of the content of the subthalamic-amygdaloid pathway in the thalamus-amygdaloid pathway NMDA receptor-mediated current and NMDA receptor-dependent LTP. NR2B The effect of H2S on the LTP and cognitive function can be eliminated by the opposite-sex blocking agent. Conclusion: H2S is enhanced by the enhancement of NR2B. The NMDA receptor function of the subunit plays an important role in enhancing the emotional memory. The study provides an experimental basis for the in-depth understanding of the physiological functions of the amygdala and the H2S,
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363

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本文編號：2495130