【作者】 余涛；

【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】 弱视是指在视觉发育敏感期,由于缺乏清晰视网膜图象刺激导致矫正视力低于正常的疾病,幼儿弱视有一定治疗效果,但成年弱视目前尚无有效治疗方法。哺乳动物包括人类出生后,视觉系统能够根据视觉环境及时调整和改变自身的神经联系和突触结构,这一改变发生的最敏感时期称为视觉发育可塑性关键期。单眼剥夺(monocular deprivation, MD)是研究哺乳动物视皮层可塑性的经典模型。以往研究认为,只有在关键期内的短期(3天)MD才能造成眼优势移动,即只有在关键期内的视皮层才具有可塑性。目前对幼年动物视皮层可塑性关键期启动和终止机制的研究证实,视皮层突触可塑性依赖于皮层抑制和兴奋神经通路的平衡。研究发现,自出生后双眼即被缝合或黑暗饲养的动物,其视皮层内GABA和NMDA受体的成熟受到严重影响,视觉发育维持在不成熟状态,抑制和兴奋神经通路平衡受到影响,从而延长了可塑性关键期。西南医院眼科实验室应用脑片膜片钳全细胞记录技术对大鼠视皮层进行研究,发现在正常大鼠可塑性关键期内,随着发育,NMDA受体相对于AMPA受体的作用逐渐减弱,双眼形觉剥夺(binocular form deprivation, BFD)通过NMDA受体与AMPA受体的相互作用影响了视皮层神经元兴奋性突触传递的功能可塑性。同时,西南医院眼科实验室对正常及BFD大鼠视皮层IV层GABA能神经元抑制性突触后电流(inhibitory postsynaptic currents,IPSCs)进行分离,发现正常大鼠GABA能抑制性回路神经元突触发育的高峰期迟于谷氨酸能兴奋性回路神经元,IPSCs峰值在成年后则保持在稳定的高水平;在可塑性关键期内,BFD抑制了大鼠GABA能抑制性突触传递功能发育。2002年Pizzorusso等的研究结果为关键期终止后成年视皮层可塑性再激活提供了直接证据,他们采用chABC酶注入大鼠颅腔,降解视皮层硫酸软骨素,成功恢复了成年大鼠视皮层眼优势移动。然而,该方法有破坏性,临床应用存在明显局限性。那么能否通过自然的、无创的异常视觉环境再激活成年视皮层可塑性呢?近期研究发现经长时间黑暗饲养的成年大鼠在短期MD后也能导致眼优势移动。这种无创方法成功再激活成年动物视皮层可塑性,提示异常视觉环境同样可影响成年视皮层,只是这些异常视觉环境需要对成年动物作用更长时间。与黑暗饲养模型比较,双眼睑缝合后所致的形觉剥夺是一种更接近临床的动物模型。既然黑暗饲养和BFD后的幼年动物能够通过影响抑制和兴奋性神经通路的成熟,使其视觉发育维持在不成熟状态,从而延长可塑性关键期,而成年大鼠的视皮层可塑性在黑暗饲养一定时间后也能被激活,那么,我们有理由假设,成年大鼠在BFD后也可能造成MD所引起的眼优势移动,而其机制可能在于调节视皮层抑制性和兴奋性神经递质受体的重新表达和分布,最终导致抑制性神经通路和兴奋性神经通路传递的失平衡,从而再激活成年视皮层可塑性。本研究拟为成年弱视的治疗提供新的思路。为此,本研究采用了以下技术和方法,探讨成年Long-Evans大鼠在BFD后,视皮层可塑性再激活的可能机制。1.采用视觉诱发电位(visual evoked potentials,VEPs)记录方法验证Long-Evans大鼠视皮层可塑性关键期结束的时间,然后采用成年大鼠进行BFD,最后选择短期MD就能引起眼优势移动的BFD时间。结果发现:(1)正常幼年大鼠(小于等于P5W)对MD的反应表现为被剥夺眼反应的早期减弱和非剥夺眼反应的缓慢增强;短期(3天)MD就能造成幼年大鼠视皮层眼优势移动。(2)MD不能造成正常成年大鼠(大于P6W)双眼反应的明显改变,也不能造成其眼优势移动。(3)BFD14天后的成年大鼠视皮层眼优势可在短期(3天)MD后发生移动,说明BFD14天组模型可以稳定地激活成年大鼠视皮层可塑性;BFD14天后的成年大鼠对MD的反应也表现为被剥夺眼反应的早期减弱和非剥夺眼反应的缓慢增强,类似于幼年大鼠对MD的反应。(4)因此在以下两部分,本研究均选择BFD 14天P7W大鼠作为成年大鼠双眼形觉剥夺后视皮层可塑性再激活实验组模型。2.采用免疫组化染色和免疫印迹技术,对P1W-P9W正常大鼠和P7W BFD 14天模型大鼠的视皮层中抑制性和兴奋性递质受体亚型分别进行标记和检测,探索大鼠视皮层中抑制性和兴奋性神经递质受体表达的发育特征以及BFD对成年视皮层内抑制性和兴奋性递质受体亚型表达和分布的影响。结果发现:(1)GABAAα1主要分布在视皮层II-III层和IV层,其在未睁眼时(P1W)少量表达,自睁眼后视皮层可塑性关键期高峰(P3W)时,表达明显增强,到可塑性关键期结束时(P5W)到高峰,至成年期(P7W)保持高水平表达,而BFD14天造成了成年大鼠视皮层GABAAα1阳性表达的减弱。(2)NMDA-NR2A主要分布在视皮层II-III层,其在未睁眼时少量表达,可塑性关键期高峰时最明显,之后表达减少至成年期维持一定水平,而BFD14天可以造成成年大鼠视皮层NMDA-NR2A阳性表达的减弱。(3)NMDA-NR2B在未睁眼时表达较明显,可塑性关键期高峰时表达最多,之后表达下降稳定在成年期低水平,而BFD14天可以造成成年大鼠视皮层NMDA-NR2B表达增强。(4)GluR-1平均分布在视皮层各层,在出生后少量表达,之后随年龄增长逐渐增强,其表达在可塑性关键期终止时达到高峰并在成年期维持较高水平,而BFD14天对成年大鼠视皮层GluR-1阳性表达没有影响。3.采用视皮层脑片膜片钳全细胞记录和电流分离技术,分别记录P1W-P9W正常大鼠和P7W BFD 14天大鼠视皮层神经元膜学特性、突触后电流、GABAA抑制性突触后电流(GABAA-IPSCs)与NMDA兴奋性突触后电流(NMDA-EPSCs),探讨视皮层神经元递质通路变化的发育特性以及BFD对成年大鼠视皮层神经元突触传递特性的影响。结果发现:(1)从睁眼前到视皮层发育可塑性关键期高峰,视皮层II-III层神经元的电学成熟度以及突触功能逐渐成熟,到关键期结束时基本稳定接近成年水平,提示视皮层II-III层神经元的电学成熟度以及突触功能是视觉经验依赖性的。而BFD不改变成年大鼠视皮层神经元的膜学特性和PSCs电学指标。(2)GABAA-IPSCs峰值随年龄的增长逐渐增大,在可塑性关键期结束时达到高峰,之后在成年期维持高水平,说明其峰值变化是视觉经验依赖性的,并且GABAA-IPSCs的复极化时间随着神经元的成熟也经历了从短到长的变化过程。BFD减弱了成年大鼠视皮层神经元GABAA-IPSCs的峰值,但对其复极化的时间没有影响。(3)NMDA-EPSCs峰值和NMDA-EPSCs在总EPSCs中所占比例在可塑性关键期高峰时占优势,之后便降低维持在一定成年期低水平,说明其发育变化是视觉经验依赖性的,但BFD对成年大鼠视皮层神经元EPSCs、NMDA-EPSCs的峰值以及NMDA-EPSCs在总EPSCs中所占比例没有明显影响。NMDA-EPSCs复极化时间随着发育是逐渐缩短的,这与NMDA受体亚型的发育变化有关,是视觉经验依赖性的,而BFD不影响成年大鼠视皮层神经元NMDA-EPSCs的复极化时间。本研究得到以下结论:1、BFD可成功再激活成年大鼠视皮层可塑性,形觉剥夺和光觉剥夺一样可以终身增强其眼优势可塑性。2、成年大鼠BFD后,视皮层GABAAα1受体表达的减少和其介导的GABA能抑制性神经回路强度的减弱可能是成年大鼠视皮层重新进入关键期的钥匙,是成年大鼠视皮层可塑性再激活的重要分子机制。3、成年大鼠在BFD后可诱导出MD所引起的眼优势移动,其机制可能在于调节视皮层抑制性和兴奋性神经递质受体的重新表达和分布,最终导致抑制性和兴奋性神经通路传递的失平衡,从而再激活视皮层可塑性。更多还原

【Abstract】 Objective: Ocular dominance (OD) plasticity is a prominent feature of the mammalian visual cortex. Brief monocular deprivation (MD) induces a rapid shift in the OD of binocular neurons in the juvenile rat visual cortex but is ineffective in adults. Although such neural changes are most evident during development, adult cortical circuits also can be modified by a variety of manipulations. Elucidating the underlying mechanisms at the cellular and synaptic levels is an essential step in understanding neural plasticity in the mature animal. 1. Visual evoked potentals (VEPs) was applied to detect the OD shift in order to build the animal model whose visual cortex plasticity was reactivated by long time’s binocular form deprivation (BFD) from adulthood. 2. Immunohistochemistry and immunoblotting were applied to investigate the changes of neural transmitter receptors in the visual cortex of adult rats manipulated by BFD. 3. Patch-clamp whole cell recording was applied to investigate the electrophysiological property of layer II-III neurons from visual cortex of adult rats manupilated by BFD. Investigation of the underlying mechanisms of how BFD reactivates OD plasticity in adult rat will help interpret the principle of adult visual cortex plasticity, and provide theoretical basis for the treatment of adult amblyopia.Methods: 1. A left craniotomy was performed over left visual cortex (centered at 7.0 mm posterior to bregma and 4 mm lateral to midline), keeping dura intact. The recording electrode was placed on the dura. The amplitude of P100 wave was used to assess the cortical response to visual stimulation. VEPs were amplified, filtered and averaged in synchrony with the stimulus. VEPs were recorded in the pigmented Long–Evans rats aged from postnatal week (PW) 3 to PW7 and recorded again after 3d, 5d or 7d’s MD in the right eyes. Rats of PW7 were treated with BFD for 7d, 10d or 14d, and then left eyes were opened for 3d, 5d or 7d befor the next VEPs recording. 2. Normal Long–Evans rats aged from PW1 to PW9 and PW7 rats after 14d’s BFD were decapitated and sections were stained with immunohistochemistry. Binocular visual cortices were dissected bilaterally after anesthetization and quantitative immunoblotting were performed. All immunoblots were performed with the experimenter blind to experimental conditions. For the immunohistochemistry and immunoblots, same primary antibodies were used, including anti-GABAA-α1, anti-NR2A, anti-NR2B and anti-GluR1. 3. The visual cortex slices were prepared from Long–Evans rats aged from PW1 to PW9 and PW7 rats after 14d’s BFD. Patch-clamp whole cell recording techniques were adopted. Pre-synaptic stimulation was given at 0.5 mA through bipolar stimulating electrodes placed in layer IV. PSCs of layer II-III neurons were recorded by recording electrodes. Then GABAA-IPSCs were isolated by holding the membrane potential at 0 mV, which is the reversal potential for EPSCs. Likewise, EPSCs were isolated by holding the membrane potential at -60 mV, which is close to the reversal potential for IPSCs. Then NMDA-EPSCs were isolated by adding CNQX into artificial cerebrospinal fluid (ACSF) after EPSCs were obtained. Above current isolations were confirmed by pharmacological method.Results: 1. Brief (3 d) MD decreased C/I ratio (contralateral VEPs amplitude/ ipsilateral VEPs amplitude to the occluded eye) in rats from PW3, PW4 and PW5 but not in those from PW6 and PW7, confirming the absence of rapid OD plasticity in adults. However, when brief MD was preceded by 14 d of BFD, we observed a significant decreasing in the C/I ratio in rats from P7W, demonstrating the reactivation of rapid OD plasticity in adult rat. 2. Studies of immunohistochemistry and immunoblotting have shown that expression of GABAA-α1 reached its peak at PW5 and BFD induces a significant decrease in the level of GABAA-α1 receptors in adult visual cortex. Expression of NR2A and NR2B reached its peak at PW3 and BFD induces a significant increase in the level of NR2A receptors and a significant decrease in the level of NR2B in adult visual cortex. Expression of GluR1 reached its peak at PW5 and BFD did change the level of GluR1 receptors in adult visual cortex. 3. Input resistance (IR), resting membrane potential (RMP) and the peak value of PSCs were not changed in adult visual cortex treated with BFD. The peak value and 10-90% decaytime of GABA-IPSCs increased with age from PW1 in normal rat visual cortex, then reached its peak at P5W and kept a high level in adulthood. BFD statistically decreased the peak value of GABA-IPSCs but not the 10-90% decaytime. The peak value of NMDA-EPSCs and the NMDA-EPSCs/EPSCs ratio increased with age since PW1 in normal rat visual cortex and then reached its peak at PW3. The 10-90% decaytime of NMDA-EPSCs decreased with age. BFD had no effect on the peak value and 10-90% decaytime of EPSCs and NMDA-EPSCs, as well as the NMDA-EPSCs/EPSCs ratio.Conclusion: 1. BFD could successfully reactivate the OD plasticity in adult visual cortex which showed a similar response as that in junvenile rats, demonstrating that form deprivation could enhance the OD plasticity of rat visual cortex for a life time. 2. The decreased expression of GABAA-α1 receptors and the decreased peak value of GABA-IPSCs in visual cortex of adult rats after BFD suggest BFD affects inhibitory synaptic transmission through GABAA receptors, which might be the key to re-enter the cortical period and the possible mechenism for the reactivation of OD plasticity in adult visual cortex. 3. Our data demonstrate the molecular changes in inhibitory neurotransmitter receptors and the induced inhibitory currents observed in the adult visual cortex that we propose enable the reactivation of rapid ocular dominance plasticity更多还原