【作者】 陆新江；

【导师】 杜继曾； 陈学群；

【作者基本信息】 浙江大学 ， 神经生物学， 2009， 博士

【摘要】 小鼠出生以后经历间歇低氧（16%O₂,4小时/天,持续4周）增强海马区LTP和空间学习记忆。学习记忆的分子机制是突触可塑,受到突触后膜功能蛋白的调节。树突棘相关的RapGAP（SPAR）可以和突触后致密物质95（PSD-95）、NMDA受体（NMDAR）形成复合物,调节树突棘的形态,可能影响学习记忆。本研究通过免疫印迹发现小鼠出生后经历间歇低氧导致发育期间生后14天（P14）、P28、P35海马区SPAR表达上调;NMDAR和PSD-95蛋白仅在P14天表达上调,而在P28和P35表达回复到对照水平。免疫荧光进一步证实,SPAR表达增强主要是在海马区富含突触的神经毡区域。小鼠海马区双侧埋管,SPAR反义核酸在注射以后第2天、第3天显著性敲低海马区SPAR表达,而在第4天,SPAR蛋白表达水平和对照没有明显差异。反义核酸处理使间歇低氧诱导空间学习记忆增强小鼠的水迷宫学习能力受到明显损伤,但是视觉敏感和游泳速度没有发生改变;而常氧发育小鼠反义核酸处理也导致空间学习记忆损伤。此外,水迷宫训练以后,SPAR反义核酸处理的小鼠记忆保持能力减弱。八臂迷宫检测显示,SPAR敲低小鼠的工作记忆也受到损伤。电生理研究发现,SPAR敲低小鼠海马脑片,基础突触传递没有受到影响,而早期LTP和晚期LTP明显减弱。上述发现说明生后间歇低氧通过上调海马区的SPAR表达增强空间学习记忆。当大脑经历应激或损伤,IGF家族蛋白在脑内表达上调。我们假设SPAR的表达受到IGF家族蛋白的调控。小鼠在出生后暴露于间歇低氧,IGF-I在4周的间歇低氧时期P9,P14,P21,P28表达上调;IGF-IR在P28和P35表达上调,IGFBP-2从P14到P35表达上调。小鼠海马区注射抗体阻断IGFBP-2的作用,发现水迷宫和没有水迷宫处理的小鼠海马区SPAR表达均下调。和IgG处理的对照小鼠相比,IGFBP-2抗体处理导致小鼠水迷宫表现损伤。水迷宫训练期间海马区IGF-I mRNA表达上调。而IGF-IR拮抗剂注射没有导致SPAR表达下调。本研究首次证实间歇低氧诱导小鼠海马区SPAR高表达参与突触可塑和空间学习记忆。间歇低氧诱导的SPAR表达上调可能来源于发育期间IGFBP-2表达上调。更多还原

【Abstract】 In our previous investigation,neonatal exposure to mild intermittent hypoxia enhances the amplitude of hippocampal long-term potentiation(LTP) and spatial learning and memory in mice,but the mechanism is still unknown.SPAR(a spine-associated RapGAP) forms a complex with PSD-95 and NMDA receptors (NMDARs) and morphologically regulates dendritic spines that may contribute to learning and memory.Here,we showed that neonatal exposure to IH uniquely led to the up-regulation of hippocampal SPAR expression during postnatal development in mice.To test whether this up-regulation is associated with the enhancement of spatial learning and memory, the dorsal hippocampi of bilaterally cannulated mice were infused with SPAR antisense, missense,or PBS.The SPAR antisense treatment time-dependently disrupted hippocampal SPAR expression.The acquisition of the Morris water maze was impaired, but visual acuity test and swimming speed were unchanged in SPAR antisense treated mice.Furthermore,the memory retention of SPAR antisense treated mice was decreased after the training of the water maze compared with controls.The working memory was also impaired in SPAR knockdown mice by measuring in the eight-arm maze.In the hippocampal SPAR knockdown mice,basal synaptic transmission was not affected,while LTP was significantly attenuated.Our finding suggests intermittent hypoxia enhances spatial learning by up-regulating the SPAR expression.IGF-I has been identified as a neurotrophic factor and the expression levels IGF family proteins are increased in brain,which undergoes stress and/or injury.The IGF-I protein expression was up-regulated during the four weeks hypoxia period;the IGF-IR was up-regulated at postnatal 28(P28) and P35;and the IGFBP-2 was up-regulated from P14 to P35 in the hippocampus of IH-treated mice compared with control. Furthermore,IGFBP-2 antibody was infused into the dorsal hippocampi of bilaterally cannulated mice.Hippocampal SPAR expression was down-regulated by IGFBP-2 antibody treatment both in water maze training-undergoing mice and without water maze training ones.Compared with IgG-treated controls,the performance of water maze was impaired in IGFBP-2 antibody-treated mice.The water maze training increased the IGF-I mRNA expression in hippocampus.However,the IGF-IR antagonist infusion could not down-regulate the expression of SPAR.This study provides the first evidence that SPAR is functionally required for synaptic plasticity and spatial learning and memory in IH treated and normoxic mice.The IH induced up-regulation of SPAR expression may result from increased expression of IGFBP-2.更多还原