【作者】 王昌鹏；

【导师】 阴正勤；

【作者基本信息】 第三军医大学 ， 眼科学， 2008， 博士

【摘要】 弱视是一种眼科常见疾病,弱视患者占世界人口的2-5%,中国有1000多万弱视患者。弱视患者的矫正视力低于相同年龄正常人的视力,同时还有对比敏感度,立体视觉,运动知觉等的损害。大量研究表明弱视主要的损害部位在视皮层,弱视的发生和治疗与视觉发育可塑性关键期密切相关,在视觉发育可塑性关键期内,如先天性白内障或斜视等异常的视觉环境可以导致弱视,在可塑性关键期内如果能及时地消除异常的视觉环境,弱视能治愈;在视觉发育可塑性关键期终止以后,异常的视觉环境则不再能引起弱视,但弱视的治疗效果明显降低,在临床上成年和大于12岁的弱视患者基本不能被治愈。因此,视觉发育可塑性关键期的终止与否对弱视治疗效果具有非常重要的意义。人类和哺乳动物出生后,视觉系统在外界环境刺激和内在基因调控下,会发生形态和功能的适应性变化,这种变化的最敏感时期称为视觉发育可塑性关键期。近年来的研究发现对视觉发育可塑性关键期终止有重要影响的因素是:视皮层内突触可塑性、局部兴奋性/抑制性神经元回路的成熟、神经元细胞外基质、神经营养因子及一些相关基因的表达。近年研究发现细胞外基质中一些成分,如硫酸软骨素蛋白多糖(chondroitin sulphate proteoglycan, CSPG)在中枢神经系统发育晚期逐渐浓缩在神经元周围,形成包围神经元胞体和树突的神经元周围网络(Perineuronal nets, PNNs)。大鼠视皮层内CSPG形成PNNs的过程在视觉发育可塑性关键期后期,暗饲养能延迟大鼠视觉发育可塑性关键期的终止,也能导致视皮层内PNNs数目显著减少,从而表明由CSPG形成的PNNs参与了视觉可塑性关键期的终止。大鼠视皮层内兴奋性突触传递主要由NMDA受体和AMPA受体介导,正常大鼠视觉发育可塑性关键期高峰后,NMDA受体电流在兴奋性突触后电流中所占比率降低,AMPA受体电流所占比率增加,暗饲养能延迟大鼠NMDA受体电流的发育性变化,提示兴奋性突触的发育变化可能也参与了大鼠视觉可塑性关键期的终止过程。这些研究结果虽然表明CSPG和兴奋性突触的发育都与大鼠视觉发育可塑性关键期终止有关,但它们参与关键期终止的具体机制仍不明确。本课题提出如下假说:在视觉发育可塑性关键期后期,视皮层内CSPG逐渐发育成熟,形成PNNs包裹在神经元胞体和树突周围,对神经元的兴奋性突触后受体具有选择性屏蔽作用。在可塑性关键期终止前后及成年阶段,PNNs选择性屏蔽NMDA受体,导致兴奋性突触传递中NMDA受体介导的成分逐渐降低,AMPA受体介导的成分逐渐增加,兴奋性突触的结构和功能逐渐达到成年的稳定状态,兴奋性突触可塑性水平降低,这些变化参与视皮层可塑性关键期终止过程。为了验证此假说,我们从以下几个方面进行研究。第一部分研究:正常大鼠视皮层可塑性关键期终止前后兴奋性突触后电流的发育变化。通过膜片钳技术,我们研究了出生后3周到8周(P3W-P8W),正常大鼠视皮层谷氨酸能兴奋性突触后电流(Glutamatergic excitatory postsynaptic current, Glu-EPSC)及其两种成分,NMDA受体电流(NMDA receptor-mediated excitatory postsynaptic current, NMDA-EPSC)和AMPA受体电流(AMPA receptor-mediated excitatory postsynaptic current, AMPA-EPSC)的发育变化。这部分结果显示:1. P3W-P8W正常大鼠视皮层神经元Glu-EPSC幅值随发育逐渐升高,P7W达峰值,P3W-P8W正常大鼠视皮层神经元NMDA-EPSC幅值随发育变化无统计学意义,P3W-P8W正常大鼠视皮层神经元AMPA-EPSC幅值随发育逐渐升高, P6W达峰值, P6W以后无显著变化。2.NMDA-EPSC/Glu-EPSC比率随发育降低, P6W时达到最小值,AMPA-EPSC/Glu-EPSC比率随发育增加,P6W时达到最大值。这些结果提示发育过程中视皮层兴奋性神经元网络受体亚型构成比的变化是导致视皮层可塑性关键期终止的机制之一。第二部分研究:降解CSPG对大鼠视皮层神经元兴奋性突触后电流的影响。通过大鼠视皮层内注射硫酸软骨素酶(chondroitinaseABC, chABC)制作成CSPG降解大鼠模型,用膜片钳技术记录了P3W-P8WCSPG降解大鼠视皮层Glu-EPSC, NMDA-EPSC, AMPA-EPSC的发育变化,并与同期正常大鼠比较,结果显示: 1. P5W-P8WCSPG降解大鼠视皮层神经元Glu-EPSC幅值显著高于同期正常大鼠,P4W-P8WCSPG降解大鼠视皮层神经元NMDA-EPSC幅值显著高于同期正常大鼠,P6W-P8WCSPG降解大鼠视皮层神经元AMPA-EPSC幅值显著高于同期正常大鼠。2. P6W-P8WCSPG降解大鼠视皮层神经元NMDA-EPSC/Glu-EPSC比率显著高于同期正常大鼠,P6W-P8WCSPG降解大鼠视皮层神经元AMPA-EPSC/Glu-EPSC比率较同期正常大鼠显著降低。3.降解CSPG后,大鼠视皮层神经元NMDA-EPSC幅值的增长百分率明显大于AMPA-EPSC幅值的增长百分率。这部分结果提示可塑性关键期后期到终止期CSPG对NMDA-EPSC具有选择性抑制作用。第三部分研究:降解CSPG对大鼠视皮层NMDA/AMPA受体表达的影响。通过免疫荧光标记技术和蛋白质印迹技术我们研究了可塑性关键期终止前后CSPG降解大鼠和正常大鼠视皮层NMDA受体和AMPA受体表达的变化。免疫荧光标记结果显示:1. P3W-P8W正常大鼠视皮层2-3层NR1阳性细胞数目无显著性变化,4层NR1阳性细胞数目在P5W以后显著降低,P5W-P8W差异不显著;P4W-P8WCSPG降解大鼠视皮层2-4层NR1阳性细胞数目显著高于同期正常大鼠。2. P3W-P8W正常大鼠视皮层2-3层GluR2阳性细胞数目随发育增加,4层GluR2阳性细胞数目在P3W-P5W期间增加,P5W-P8W差异不显著。P6W-P8WCSPG降解大鼠视皮层2-3层GluR2阳性细胞数目显著高于同期正常大鼠,P7W-P8WCSPG降解大鼠视皮层4层GluR2阳性细胞数目显著高于同期正常大鼠。3.降解CSPG以后,大鼠视皮层2-4层NR1阳性细胞数目的增长百分率大于GluR2阳性细胞数目的增长百分率。蛋白质印迹技术的结果显示: 1. P3W-P4W正常大鼠视皮层NR1表达水平较高,然后逐渐降低,P3W-P4W与P7W-P8W之间差异具有统计学意义,降解CSPG后,P4W-P8WCSPG降解大鼠视皮层NR1的表达水平显著高于同期正常大鼠。2.正常大鼠GluR2表达水平在P3W-P6W随发育逐渐升高,P6W-P8W之间差异无统计学意义;降解CSPG后,P6W-P8WCSPG降解大鼠视皮层GluR2表达水平显著高于同期正常大鼠。这些结果表明:从视觉可塑性关键期后期到终止期,视皮层神经元NMDA受体是存在的,CSPG选择性抑制NR1蛋白的表达水平而屏蔽了神经元上NMDA受体与递质的结合;同时,提示CSPG对视皮层神经元AMPA受体的影响只表现在成年期。综上所述,本研究可以得到以下结论:从视觉可塑性关键期高峰到终止期,CSPG形成的PNNs选择性地“屏蔽”了NMDA受体的表达和功能,导致AMPA受体的表达和功能相对增强,AMPA受体在谷氨酸能兴奋性突触传递中发挥优势作用,兴奋性突触被增强和固化,突触可塑性降低,可塑性关键期终止。因此CSPG形成的PNNs选择性“屏蔽”谷氨酸能兴奋性突触中NMDA受体,导致了视皮层兴奋性神经元网络NMDA受体和AMPA受体构成比的变化。大鼠视皮层兴奋性神经元网络NMDA受体和AMPA受体构成比的变化是视皮层可塑性关键期终止的机制之一。更多还原

【Abstract】 Amblyopia is a common disease in ophthalmology. In world, 2-5% population are amblyopia patients including more than 10 million amblyopia children in China. Amblyopia patients have correct acuity lower than that of normal person at same age, impaired contrast sensitivity, stereoscopic vision and motion perception. Many studies suggest that visual cortex is the crucial impaired target of amblyopia. Development and therapy of amblyopia have relation with the critical period of visual plasticity. During critical period of visual plasticity, abnormal visual experience, such as congenital cataract and strabilismus, can lead to amblyopia and amblyopia can be cured after abnormal visual experience removed. After the end of critical period of visual plasticity, abnormal visual experience can not lead to amblyopia and amblyopia developed previously can not be cured. Clinically adult amblyopia patients and amblyopia children elder than 12 year can not be cured. Therefore whether the critical period of visual plasticity end or not has very important significance for therapeutic efficacy of amblyopia.Development of the visual cortex is strongly modified by visual experience during early short period of postnatal development called critical period of visual plasticity. Recently investigations suggest that factors have effects on the end of critical period of visual plasticity, including synaptic plasticity in visual cortex, maturation of excitatory circuit and inhibitory circuit, extracellular matrix, neurotrophic factors and expression of related gene. Recent studies found that chondroitin sulphate proteoglycan(CSPG), a component of extracellular matrix, condensed around a group of neurons and formed perineuronal nets(PNNs) encapsuling the soma and dendrites of neurons in central nervous system. The PNNs was formed with CSPG during the late phase of critical period. Dark rearing delay the end of critical period of visual plasticity, also decrease the number of PNNs, suggesting that the PNNs formed with CSPG is involved in the end of critical period of visual plasticity.In rat visual cortex, glutamatergic excitatory transmission is mediated by NMDA receptor and AMPA receptor. After the peak of critical period of rat visual plasticity, the proportion of NMDA receptor mediated excitatory postsynaptic current(NMDA-EPSC) in glutamatergic excitatory postsynaptic current(Glu-EPSC) gradually decrease and the proportion of AMPA receptor mediated excitatory postsynaptic current(AMPA-EPSC) in Glu-EPSC gradually increase. Dark rearing can delay developmental changes in properties of NMDA-EPSC in rat visual cortex, implying that developmental changes in Glu-EPSC might be involved in the end of critical period of visual plasticity. These investigations suggest that developmental changes in CSPG and excitatory synapses have relation with the end of critical period of visual plasticity. However mechanisms ending the critical period are not well known.We hypothesized that: During late phase of critical period, the PNNs formed with CSPG encapsule the soma and dendrites of neurons in visual cortex, and preferentially“shield”NMDA receptor. This leads to NMDA receptor mediated component in excitatory transmission decrease, and AMPA receptor mediated component in excitatory transmission increase, which result in the plasticity of excitatory synapses decrease gradually and the end of critical period of visual plasticity. To identify this hypothesis, the present study was performed.Part 1. Developmental changes in glutamatergic excitatory postsynaptic current in normal rat visual cortex around the end of cirtical period.Using patch clamp technique, we studied developmental changes in Glu-EPSC, NMDA-EPSC and AMPA-EPSC in normal rat visual cortex from P3W to P8W. Results in this study suggested that: 1. The NMDA-EPSC amplitude had no significant changes from P3W to P8W, whereas amplitudes of Glu-EPSC and AMPA-EPSC increased gradually from P3W to P8W, achieved peak value at P7W and P6W, respectively. 2. The NMDA-EPSC/Glu-EPSC ratio decreased gradually and achieved lowest level at P6W, whereas the AMPA-EPSC/Glu-EPSC ratio increased gradually and achieved peak level at P6W. These results indicated that developmental changes in NMDA-EPSC/Glu-EPSC ratio and AMPA-EPSC/Glu-EPSC ratio might be one of mechanisms ending the critical period of visual plasticity.Part 2. Effects of degradating CSPG on Glu-EPSC, NMDA-EPSC and AMPA-EPSC in rat visual cortexCSPG degradation rats were made by normal rats visual cortex chondroitinase ABC(chABC) injection. Developmental changes in Glu-EPSC, NMDA-EPSC and AMPA-EPSC in CSPG degradation rat visual cortex were studied by patch clamp technique. To explore the effect of CSPG on developmental changes in Glu-EPSC, NMDA-EPSC and AMPA-EPSC in rat visual cortex, data from CSPG degradation rats were compared with those from normal rats. Results in this study suggested that: 1. NMDA-EPSC amplitudes of CSPG degradation rats were significant higher than those of normal rats from P4W to P8W. AMPA-EPSC amplitudes of CSPG degradation rats were significant higher than those of normal rats from P6W to P8W. 2.From P6W to P8W, the NMDA-EPSC/Glu-EPSC ratio of CSPG degradation rat was significant higher than that of normal rat, whereas the AMPA-EPSC/Glu-EPSC ratio of CSPG degradation rat was significant lower than that of normal rat. 3. After CSPG degradated, the increase percentage of NMDA-EPSC amplitude was higher than that of AMPA-EPSC amplitude in rat visual cortex. These results suggested that CSPG preferentially had inhibitory effect on the NMDA-EPSC amplitude in rat visual cortex before the end of critical period.Part 3. Effects of degradating CSPG on expression of NMDA receptor and AMPA receptor in rat visual cortexUsing immunofluorescence, we investigated developmental changes in NMDA receptor subunit NR1 immunoreactive(NR1-IR) neurons and AMPA receptor subunit GluR2 immunoreactive(GluR2-IR) neurons in visual cortex of CSPG degradation rat and normal rat. Results in this study suggested that: 1. The number of NR1-IR neurons in normal rat visual cortex layer 2-3 had no significant changes from P3W to P8W. The number of NR1-IR neurons in normal rat visual cortex layer 4 decreased from P3W to P5W, and had no significant changes from P5W to P8W. The number of NR1-IR neurons in visual cortex layer 2-4 of CSPG degradation rat was significant higher than that of normal rat from P4W to P8W. 2. The number of GluR2-IR neurons in normal rat visual cortex layer 2-3 gradually increased from P3W to P8W,whereas the number of GluR2-IR neurons in normal rat visual cortex layer 4 gradually increased from P3W to P5W and had no significant changes from P5W to P8W. The number of GluR2-IR neurons in CSPG degradation rat visual cortex layer 2-3 was higher than that in normal rat visual cortex from P6W to P8W. The number of GluR2-IR neurons in CSPG degradation rat visual cortex layer 4 was higher than that in normal rat visual cortex from P7W to P8W. 3. After CSPG degradated, the increase percentage of NR1-IR neurons in rat visual cortex layer 2-4 was higher than that of GluR2-IR neurons. Using western blot, we investigated developmental changes in NR1 level and GluR2 level in visual cortex of CSPG degradation rat and normal rat. Results analyzed by western blot suggested that: 1. NR1 level in normal rat visual cortex decreased gradually. NR1 level in normal rat visual cortex from P3W to P4W were higher that those from P7W to P8W. NR1 level in CSPG degradation rat visual cortex was significant higher than that in normal rat visual cortex from P4W to P8W. 2. GluR2 level in normal rat visual cortex increased gradually from P3W to P6W and had no significant changes from P6W to P8W, whereas GluR2 level in CSPG degradaton rat visual cortex was higher than that in normal rat visual cortex from P6W to P8W.These results indicated that from the late phase to the end of critical period of visual plasticity, NMDA receptor had been present, whereas expression of NR1 protein was“shield”preferentially by PNNs formed with CSPG. This blocked the combination of transmitter and NMDA receptor. In addition, these results indicated that CSPG had only effects on AMPA receptor during adulthood.Summarized results in present study, it could be concluded that from the peak to the end of critical period, the PNNs formed with CSPG preferentially“shield”expression and function of NMDA receptor in rat visual cortex, leading to relatively enhancement in expression and function of AMPA receptor. This results in that AMPA receptor had predominate role in excitatory transmission around the end of critical period. These changes in NMDA receptor and AMPA receptor lead to that glutamatergic excitatory synapses were strengthened, synaptic plasticity decreased and critical period closured. Thus the PNNs formed with CSPG preferentially“shield”NMDA receptor in rat visual cortex lead to changes in constituent ratio of NMDA receptor and AMPA receptor in excitatory neuronal network in rat visual cortex that might be one of mechanisms ending critical period of visual plasticity.更多还原