【摘要】： 在外界環(huán)境刺激和內(nèi)在基因調(diào)控下,視覺系統(tǒng)可以發(fā)生形態(tài)和功能的適應性改變,這種發(fā)育的敏感時期稱為視覺發(fā)育可塑性關(guān)鍵期(Critical period, CP)。在生后早期的關(guān)鍵期內(nèi),視覺經(jīng)驗可以促進突觸之間的連接及中樞神經(jīng)元網(wǎng)絡系統(tǒng)的成熟。異常視覺環(huán)境下,如斜視、上瞼下垂或先天性白內(nèi)障等的存在,使兒童視網(wǎng)膜缺乏正常清晰的圖像刺激而導致矯正視力低于正常值。弱視患者還往往伴有立體視覺、運動、知覺等的損害。如果在可塑性關(guān)鍵期內(nèi)及時排除這些影響,弱視可以治愈,而在可塑性關(guān)鍵期終止以后,即使獲得正常的視覺輸入,弱視的治愈機率也會很低,因為,人類的視皮層可塑性隨著關(guān)鍵期的終止被抑制了。在臨床上,成年和大于12歲的兒童弱視患者基本不能被治愈。已經(jīng)證明,弱視的發(fā)病部位位于視皮層。所以,如何啟動被抑制的視皮層可塑性,是解決問題的關(guān)鍵。 新近研究發(fā)現(xiàn),對視覺發(fā)育可塑性關(guān)鍵期終止有重要影響的因素是:視皮層內(nèi)突觸可塑性、局部興奮性/抑制性神經(jīng)元回路的成熟、神經(jīng)元細胞外基質(zhì)(extracellular matrix, ECM)、神經(jīng)營養(yǎng)因子以及一些相關(guān)基因的表達。細胞外基質(zhì)中的一些成分,如硫酸軟骨素粘多糖(Chondroitin solfate proteoglycans, CSPGs),在中樞神經(jīng)系統(tǒng)發(fā)育晚期逐漸濃縮成高密度的格子狀結(jié)構(gòu),形成包圍神經(jīng)元胞體和樹突的神經(jīng)元周圍網(wǎng)絡(Perineuronal nets, PNNs),將視皮層神經(jīng)元完全套入其中。Pizzorusso等人利用硫酸軟骨素酶(chondroitinaseABC, chABC)降解成年大鼠視皮層中的CSPGs,恢復了單眼視覺剝奪成年鼠視皮層眼優(yōu)勢柱可塑性,說明CSPGs在視皮層可塑性中發(fā)揮了強大的抑制作用。而且暗飼養(yǎng)可以導致視皮層內(nèi)PNNs數(shù)目顯著減少,表明CSPGs參與形成的PNNs介入了視覺可塑性關(guān)鍵期的終止。既往研究亦發(fā)現(xiàn),視皮層內(nèi)興奮性突觸傳遞主要由N-甲基-D天冬氨酸受體(N-mehyl-D-aspartate, NMDA)及α-氨基-3羥基-5-甲基-4-異惡唑丙酸受體(a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA)介導。本實驗室在之前的研究中,應用腦片膜片鉗全細胞記錄技術(shù)對大鼠視皮層進行研究發(fā)現(xiàn):在正常大鼠可塑性關(guān)鍵期內(nèi),NMDA受體的作用相對于AMPA受體來說隨著發(fā)育逐漸減弱,其中,NMDA受體介導的興奮性突觸傳遞特性與視皮層可塑性的變化相關(guān)。還有研究發(fā)現(xiàn),NMDA受體亞單位NR2A( N-mehyl-D-aspartate 2A receptor, NR2A)、NR2B (N-mehyl-D-aspartate 2B receptor, NR2B)與視皮層可塑性密切相關(guān),且NR2A/2B比率隨發(fā)育逐漸增高,參與了視皮層可塑性的終止。通過形覺剝奪(Binocular form deprivation, BFD)或暗飼養(yǎng)可以使之出現(xiàn)降低,視皮層可塑性得到一定程度的恢復。 隨著哺乳動物由幼年期向成熟期的發(fā)育,視皮層可塑性被抑制,同時,NMDA受體功能和CSPGs發(fā)育成熟均參與可塑性關(guān)鍵期的終止,兩者之間可能具有相互聯(lián)系。但是,降解視皮層CSPGs從而再激活視皮層可塑性的過程是否通過其對NMDA受體亞單位表達的影響來實現(xiàn)的呢?目前尚不清楚。多數(shù)研究集中在大鼠幼年期和成年期NR2A、NR2B的表達變化,而對于視皮層可塑性關(guān)鍵期終止前后這一時間段內(nèi)NR2A、NR2B的表達和視皮層各層分布的研究甚少。為此,提出如下假設:在視皮層發(fā)育可塑性關(guān)鍵期終止前后,視皮層內(nèi)NR2A、NR2B參與視皮層可塑性過程,降解大鼠視皮層細胞外基質(zhì)中的CSPGs后,視皮層內(nèi)NR2A、NR2B的表達發(fā)生變化。這一受體亞單位表達的變化可能是降解視皮層CSPGs恢復視皮層被抑制的可塑性的重要分子機制之一。為了驗證此假說,我們從以下兩個方面進行研究。 第一部分:視覺發(fā)育可塑性關(guān)鍵期終止前后正常大鼠視皮層NMDA受體亞單位NR2A和NR2B的表達 通過免疫熒光標記技術(shù)和蛋白質(zhì)印跡技術(shù),我們研究了出生后3到7周正常大鼠視皮層中NMDA受體亞單位NR2A、NR2B的發(fā)育變化。本部分研究結(jié)果發(fā)現(xiàn):1.生后3-7周正常大鼠視皮層中NR2A定位在陽性細胞的胞膜和胞漿,在視皮層各層中均有明顯表達,且在視皮層II-III層相對較集中分布。生后3-5周視皮層各層中陽性細胞數(shù)及總蛋白量逐漸增高(P0.05),5-7周增高不明顯。2.生后3-7周正常大鼠視皮層中NR2B定位在陽性細胞的胞膜和胞漿,在視皮層各層中均有明顯表達,但在各層間的分布并無明顯差異;且在生后3周時各層陽性細胞數(shù)及總蛋白量表達水平最高,在生后3-6周其表達明顯降低(P0.05),6-7周表達趨近一較穩(wěn)定水平。3.通過對視皮層各層NMDA受體NR2A和NR2B表達的陽性細胞數(shù)及蛋白免疫印跡結(jié)果進行分析得出,隨大鼠年齡增長(生后3-7周),NR2A/2B比率逐漸增加,且在生后3-6周比率增加幅度有統(tǒng)計學意義(P0.05)。 第二部分:視覺發(fā)育可塑性關(guān)鍵期終止前后降解大鼠視皮層硫酸軟骨素對NR2A和NR2B表達的影響 本實驗參照Pizzorusso等的動物模型方法、利用免疫熒光標記和蛋白質(zhì)印跡技術(shù),率先研究可塑性關(guān)鍵期終止前后CSPGs降解大鼠視皮層中NMDA受體亞單位NR2A、NR2B表達的變化。研究結(jié)果發(fā)現(xiàn):1.降解大鼠視皮層中的CSPGs后,與正常組大鼠同時間點比較,視皮層II-III、V-VI層中NR2A陽性細胞數(shù)在生后4-7周有顯著性差異,而視皮層IV層在生后5-7周有顯著性差異,同時蛋白免疫印跡檢測也表明視皮層中NR2A總蛋白量在生后4-7周出現(xiàn)顯著性降低(P0.05);2.降解大鼠視皮層中的CSPGs后,NR2B的陽性細胞數(shù)及蛋白表達水平在生后各時間點較正常組大鼠均無顯著變化;3.降解大鼠視皮層中的CSPGs后,視皮層中NR2A/2B比率在生后4-7周較正常組大鼠同時間點表達比率出現(xiàn)降低,有顯著性差異(P0.05)。 由上述結(jié)果初步得出以下結(jié)論:1.在視覺可塑性關(guān)鍵期終止前后,正常大鼠視皮層中NMDA受體亞單位NR2A和NR2B參與了視皮層可塑性變化的過程,而且隨著大鼠生后周齡的增加(生后3-7周),兩者比率逐漸升高, NR2A的表達逐漸取代了NR2B的主導地位。2.降解大鼠視皮層中的CSPGs后,NMDA受體亞單位NR2A/2B表達的比率在生后4-7周出現(xiàn)降低,并且以NR2A表達的減少為主,這一受體亞單位構(gòu)成變化可能是降解視皮層CSPGs后恢復視皮層可塑性的重要分子機制之一。
[Abstract]:During the critical period of postnatal development, visual experience promotes synaptic connections and central neural network systems. Maturity. Abnormal visual conditions, such as strabismus, ptosis or congenital cataract, make the retina of children lack normal and clear image stimulation and lead to corrected visual acuity below normal. Amblyopia patients are often accompanied by stereopsis, movement, perception and other damage. If these effects are eliminated in a timely manner during the critical period of plasticity, weakness. Visual cortex plasticity in humans is suppressed with the termination of the critical period. Clinically, amblyopia in adults and older than 12 years of age is virtually incurable. It has been shown that the onset of amblyopia is limited. The location is located in the visual cortex. Therefore, how to start the inhibition of the plasticity of the visual cortex is the key to solve the problem.
Recent studies have found that the key factors affecting the termination of visual development plasticity are synaptic plasticity in the visual cortex, maturation of local excitatory/inhibitory neuronal circuits, expression of extracellular matrix (ECM), neurotrophic factors and some related genes, and some of the components of extracellular matrix. Chondroitin sulfate mucopolysaccharides (CSPGs), such as chondroitin solfate proteoglycans (CSPGs), gradually condense into high-density lattice-like structures in the late development of the central nervous system, forming perineuronal nets (PNNs), which surround the cell bodies and dendrites of neurons, and completely encapsulate the neurons in the visual cortex. Chondroitinase ABC (chABC) degraded CSPGs in the visual cortex of adult rats, restored the plasticity of dominant columns in the visual cortex of monocular deprived adult rats, suggesting that CSPGs played a powerful inhibitory role in the plasticity of visual cortex, and the number of PNNNs in the visual cortex was significantly reduced by dark feeding, suggesting that CSPGs participated in the formation of visual cortex. Previous studies have also shown that excitatory synaptic transmission in the visual cortex is mainly mediated by N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid (AMPA). In previous studies, whole-cell patch-clamp recording techniques were used to study the visual cortex in rats. It was found that the role of NMDA receptors decreased with the development of AMPA receptors during the critical period of plasticity in normal rats. Other studies have found that NR2A (N-mehyl-D-aspartate 2A receptor, NR2A) and NR2B (N-mehyl-D-aspartate 2B receptor, NR2B) are closely related to visual cortex plasticity, and NR2A/2B ratio increases with development, which participates in the termination of visual cortex plasticity. Feeding can make it appear lower, and the plasticity of the cortex is restored to some extent.
With the development of mammals from infancy to maturity, the plasticity of visual cortex is inhibited. At the same time, both NMDA receptor function and CSPGs are involved in the termination of the critical stage of plasticity, which may be interrelated. However, whether the process of degradation of visual cortex CSPGs and reactivation of visual cortex plasticity through its subunit to NMDA receptor may be related to each other. Most studies focused on the changes of NR2A and NR2B expression in young and adult rats, but few studies focused on the expression of NR2A and NR2B and the distribution of NR2B in the visual cortex before and after the termination of the critical period of visual cortex plasticity. NR2A and NR2B in the visual cortex participate in the plasticity process of the visual cortex before and after the termination of the critical period of plasticity. After the degradation of CSPGs in the extracellular matrix of the visual cortex, the expression of NR2A and NR2B in the visual cortex changes. The change of NR2A and NR2B expression may be an important molecular mechanism for the degradation of CSPGs in the visual cortex to restore the inhibited plasticity of the visual cortex. In order to test this hypothesis, we study the following two aspects.
Part I: Expression of NMDA receptor subunits NR2A and NR2B in the visual cortex of normal rats before and after termination of the critical period of visual plasticity
The development of NMDA receptor subunits NR2A and NR2B in the visual cortex of normal rats from 3 to 7 weeks after birth was studied by immunofluorescence labeling and Western blotting. The number of positive cells and total protein in each layer of visual cortex gradually increased at 3-5 weeks postnatal (P 0.05), but not at 5-7 weeks postnatal (P 0.05). 2. NR2B was localized in the membrane and cytoplasm of positive cells in the visual cortex of normal rats at 3-7 weeks postnatal, and was obviously expressed in all layers of visual cortex, but in different layers of visual cortex. There was no significant difference in the number of positive cells and the expression of total protein in different layers at 3 weeks after birth. The expression of positive cells and total protein decreased significantly at 3-6 weeks after birth (P 0.05), and reached a stable level at 6-7 weeks. The NR2A/2B ratio increased gradually with the age of rats (3-7 weeks after birth), and the increase of NR2A/2B ratio at 3-6 weeks after birth was statistically significant (P 0.05).
Part 2: Effect of chondroitin sulfate on the expression of NR2A and NR2B in visual cortex of rats before and after the termination of critical period of visual development plasticity
Referring to Pizzorusso's animal model, immunofluorescence labeling and protein imprinting were used to study the changes of the expression of NMDA receptor subunits NR2A and NR2B in the visual cortex of rats before and after the termination of the critical period of plasticity. The number of NR2A-positive cells in visual cortex II-III and V-VI was significantly different at 4-7 weeks after birth, while the number of NR2A-positive cells in visual cortex IV was significantly different at 5-7 weeks after birth. The total protein content of NR2A in visual cortex decreased significantly at 4-7 weeks after birth (P 0.05). The expression of NR2A/2B in the visual cortex was significantly lower than that in the normal group at 4-7 weeks after birth (P 0.05).
The results are as follows: 1. Before and after the termination of the critical period of visual plasticity, the NMDA receptor subunits NR2A and NR2B in the visual cortex of normal rats participated in the process of visual cortex plasticity, and the ratio of NR2A and NR2B increased gradually with the increase of postnatal age (postnatal 3-7 weeks). 2. After degradation of CSPGs in the visual cortex of rats, the expression ratio of NR2A/2B, a NMDA receptor subunit, decreased at 4-7 weeks postnatal, and the expression of NR2A was mainly decreased. The change of this receptor subunit composition may be one of the important molecular mechanisms of restoring visual cortex plasticity after degradation of CSPGs in the visual cortex.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R77

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