气体信号分子对边缘系统介导的大鼠记忆功能的影响及机制

发布时间：2019-06-08 07:57

【摘要】：第一部分气体信号分子NO对可卡因戒断所致的大鼠成瘾记忆的影响及机制目的：一氧化氮(NO)作为一种内源性气体信号分子,在中枢神经系统发挥广泛的生物学效应。有研究报道NO参与可卡因等药物成瘾的形成过程。然而,NO是否也在可卡因成瘾的戒断行为中发挥重要作用则尚未见报道。近年来的研究发现,可卡因成瘾戒断会导致伏隔核(NAc)区突触表面的a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid受体(AMPA受体)受体表达上调,而这种上调被认为与可卡因的行为敏化的维持有关。NO可通过其亚硝基化作用调节突触部位AMPA受体的膜表达。因此,本实验研究NO在可卡因成瘾戒断所致大鼠成瘾记忆中的作用及其机制。方法：采用蛋白交联的western blotting技术研究大鼠NAc区AMPA受体亚基(GluR1和GluR2)在突触部位的膜表达情况；采用脑片膜片钳技术和场电位记录方法研究NAc区AMPA受体电流和LTD的变化情况；采用自发活动及糖水偏好实验观察可卡因成瘾戒断所致的大鼠行为学变化。结果：可卡因戒断导致大鼠NAc区突触部位GluR1和GluR2亚基的蛋白表达出现不成比例的上调,表现为突触表面GluR2的表达相对较少、钙通透性AMPA受体介导的电流增加。进一步研究发现,戒断后NAc区nNOS的表达、NO的含量,以及N-ethylmaleimide-sensitive factor (NSF)的亚硝基化水平均有所增加,提示可卡因戒断所致的NAc突触表面GluR2亚基表达相对较少可能是因NSF功能不足所致。外源性使用NO供体可增加戒断大鼠NAc区的NSF亚硝基化水平并选择性上调突触部位GluR2的含量,从而恢复GluR1和GluR2膜表达平衡及钙通透性AMPA受体电流。特异性干扰NSF-GluR2结合的生物肽可阻断NO的这种效应,提示NO的作用依赖于NSF与GluR2亚基的结合。场电位实验也证实NO是通过促进NSF亚硝基化并上调GluR2的膜表达,进而逆转戒断后NAc区的突触可塑性变化(长时程抑制,LTD)。另外,外源性给予NO供体和亚硝酸钠均可通过增加NAc区NSF与GluR2的结合能力来阻断戒断后大鼠的行为敏化。特异性阻断NAc区GluR2亚基内化过程也可减轻戒断大鼠的敏化行为,提示突触表面GluR2表达相对不足是行为敏化的重要原因。结论：可卡因戒断引起的NAc区NSF亚硝基化水平和突触部位GluR2亚基表达增加是大脑出现的一种内源性代偿机制,从而限制机体对可卡因的长时程异常行为反应。靶向性调控NAc区NO的生成及NSF的硝基化水平可能是可卡因成瘾治疗的新策略。第二部分气体信号分子H2S对大鼠海马区介导的学习记忆的影响及机制目的：硫化氢(H2S)是一种内源性气体信号分子,在生物体内发挥着广泛的生物学效应。中枢神经系统的H2S作为一个突触调节分子,具有神经保护作用。近来有研究证实,神经退行性疾病(如阿尔茨海默病、帕金森病等)患者或动物大脑H2S含量出现异常。H2S可易化突触传递的长时程增强(LTP),但目前对于H2S与学习记忆的关系则尚未有相关的报道。本实验旨在研究气体信号分子H2S在大鼠海马介导的记忆及突触可塑性形成的作用及机制。方法：采用亚甲基蓝法检测大鼠海马组织H2S含量；条件性恐惧记忆和新事物认知实验观察大鼠的认知功能；脑片膜片钳和场电位技术记录大鼠海马区NMDA受体介导的电流及LTP; Western blotting实验研究大鼠海马与突触可塑性密切相关蛋白的表达水平。结果：条件性惊恐学习训练可明显增加大鼠海马区H2S的含量。H2S合成酶抑制剂可阻断训练引起的海马H2S含量增加,并损伤海马依赖的情景型恐惧记忆。外源性补充H2S可剂量依赖性的增强大鼠情景型恐惧记忆。同样,抑制内源性H2S合成可损伤大鼠海马依赖的新物体认知功能,而外源性H2S可促进动物的这种认知能力。电生理实验发现,H2S选择性增强海马区含NR2A亚基的NMDA受体介导的电流和NMDA受体依赖的海马LTP；且NR2A特异性阻断剂可取消H2S对LTP和认知功能的增强作用。Western blotting结果表明H2S的促认知作用与PKA、PKC、CaMKII和CREB等信号通路激活有关。结论：作为一种内源性气体信号分子,H2S在海马依赖记忆和突触可塑性形成过程中扮演着重要角色。该结果进一步加深了我们对H2S生理功能的理解,同时也提示H2S释放药物可能是认知障碍治疗的一种新策略。第三部分气体信号分子H2S对大鼠杏仁核区介导的情感记忆的影响及机制目的：杏仁核是产生、识别和调节情绪的关键脑区,控制情感性学习及记忆行为。有研究发现,额颞叶退变性疾病和阿尔茨海默病患者可出现杏仁核依赖的情感记忆异常；孤独症患儿的大脑杏仁核也存在功能障碍。因此,研究内源性物质在杏仁核中的功能具有重要意义。H2S作为一种内源性气体信号分子,在海马区扮演着神经调质的角色。但其是否也在杏仁核介导的情感记忆行为和突触可塑性中发挥重要作用则尚未见相关报道。方法：采用亚甲基蓝法检测大鼠脑组织H2S的含量；条件性恐惧记忆和条件性味觉厌恶实验观察H2S对大鼠情感记忆行为的影响；脑片膜片钳和场电位技术记录大鼠杏仁核区NMDA受体介导的电流及LTP; Western blotting和Golgi染色技术研究大鼠杏仁核区突触结构情况。结果：线索型恐惧学习训练增加大鼠脑组织的H2S含量。外源性给予H2S供体可明显增强杏仁核介导的线索型恐惧记忆,且这种效应在训练后3周仍持续存在。此外,H2S可减缓恐惧记忆的消散,促进恐惧记忆自发性的恢复和复发。外源性给予H2S也可明显增强杏仁核依赖的条件性味觉厌恶记忆。免疫组化实验发现H2S合成酶胱硫醚-β-合成酶(CBS)在杏仁核区有大量表达,杏仁核内微量注射CBS抑制剂可损伤大鼠的线索型恐惧记忆。进一步实验发现,H2S可增加杏仁核区神经元的树突棘密度,增加CREB活性和BDNF含量,并促进NR2B亚基的膜表达。电生理实验发现,H2S可选择性增强丘脑-杏仁核通路含NR2B亚基的NMDA受体所介导的电流及NMDA受体依赖性LTP. NR2B特异性阻断剂可取消H2S对LTP和认知功能的促进作用。结论：H2S通过增强含NR2B亚基的NMDA受体功能,从而起到增强情感记忆的作用。该研究为深入理解杏仁核及H2S的生理功能提供了实验依据,也为将H2S释放药物开发成为治疗情感障碍性疾病的药物提供新思路。
[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,
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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