双眼形觉剥夺可影响视皮质

doi:10.3969/j.issn.1673-5374.2012.34.009

摘要/Abstract

摘要：

α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体对发展期视觉皮质突触可塑性具有重要作用。实验在大鼠出生14d缝合眼皮建立双眼形觉剥夺模型，结果发现在视觉发育过程中，α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体介导的兴奋性突触后电流下降时间随发育变长，峰值随发育增大，而双眼形觉剥夺后α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体介导的兴奋性突触后电流下降时间及峰值无明显变化；但2种情况下α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体介导的兴奋性突触后电流上升时间均不随发育而变化。因此，双眼形觉剥夺影响α-氨基-3-羟基-5-甲基-4-异恶唑丙酸电流特性，形觉刺激在视皮质神经元经验依赖性修饰中发挥作用。

关键词: α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体, 兴奋性突触后电流, 全细胞记录, 视觉皮质, 双眼形觉剥夺, 视觉发育, 神经发育, 再生 , 神经再生

Abstract:

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors are considered to play a crucial role in synaptic plasticity in the developing visual cortex. In this study, we established a rat model of binocular form deprivation by suturing the rat binocular eyelids before eye-opening at postnatal day 14. During development, the decay time of excitatory postsynaptic currents mediated by α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid receptors of normal rats became longer after eye- opening; however, the decay time did not change significantly in binocular form deprivation rats. The peak value in the normal group became gradually larger with age, but there was no significant change in the binocular form deprivation group. These findings indicate that binocular form deprivation influences the properties of excitatory postsynaptic currents mediated by α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat visual cortex around the end of the critical period, indicating that form stimulation is associated with the experience-dependent modification of neuronal synapses in the visual cortex.

Key words: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, excitatory postsynaptic currents, whole-cell recording, visual cortex, binocular form deprivation, visual development, neural development, regeneration, neural regeneration

. 双眼形觉剥夺可影响视皮质[J]. 中国神经再生研究(英文版), 2012, 7(34): 2713-2718.

Mingming Liu, Chuanhuang Weng, Hanping Xie, Wei Qin . Binocular form deprivation influences the visual cortex[J]. Neural Regeneration Research, 2012, 7(34): 2713-2718.

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引言

材料方法

Design

This was a randomized, controlled, animal study.

Time and Setting

Experiments were performed in the Laboratory of Southwest Eye Hospital from August 2010 to May 2011.

Materials

A total of 40 healthy specific pathogen-free Long Evans rats were supplied by the Experimental Animal Center of Daping Hospital, Third Military Medical University of Chinese PLA (animal license No. SCXK 2007-0005) at postnatal 2–7 weeks, irrespective of sex.The rats were reared in a light-controlled room before the start of experiments, with a fixed lighting schedule (8:00 to 20:00). Light was generated by two fluorescent lamps with approximately 90 lx of intensity at the animal level[10, 18-19]. The room temperature was controlled at 22–25°C, and humidity at 50–60%[20-21]. All experimental protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China[22].

Methods

Establishment of binocular form deprivation rats

At PD 14, before eye-opening, binocular form deprivation Long Evans rats were established by suturing the binocular eyelids under sodium pentobarbital anesthesia[5, 23]. The binocular form deprivation group was inspected twice a day for holes in the sutured eyelids. We immediately repaired the holes, if necessary. Closed binocular eyelids after suturing indicated successful binocular form deprivation model establishment[24].

Preparation of rat visual cortical slices

Visual cortical slices were prepared from normal rats at PD 14, 21 (7 days after eye-opening), 35 and 49, as described previously[10, 25], and from binocular form deprivation rats at PD 21, 35 and 49. Animals were initially anesthetized with ether[26] and then decapitated. Brains were rapidly removed and placed into cold (4°C) carbogenated artificial cerebrospinal fluid, supplemented with 124 mM NaCl, 26 mM NaHCO3, 3 mM KCl, 1.25 mM NaH2PO4, 1.3 mM MgCl2, 2 mM CaCl2,10 mM dextroglucose, pH 7.4[27-28]. Subsequently, visual cortical slices[29-31] (300 μm) were dissected using a vibroslicer (Ted Pella Instrument, CA, USA), and then incubated at room temperature for at least 1 hour in carbogenated artificial cerebrospinal fluid.

Electrophysiological recording

For electrophysiological recordings, slices were transferred to a submerged-type recording chamber and continually perfused with carbogenated artificial cerebrospinal fluid (1.5 mL/min) at 20–25°C[32]. Pipettes were pulled from borosilicate glass capillaries (World Precision Instruments, USA) with the use of a P-97 micropipette puller (Sutter Instrument, Novato, USA). The electrodes had tip impedances of 5–10 MΩ when filled with a solution containing 140 mM K+ gluconate, 2 mM MgCl2, 1.1 mM ethyleneglycol-bis(β-aminoethyl ether)-n, n’-tetraacetic acid, 0.1 mM CaCl2, 10 mM hydroxyethyl piperazine ethanesulfonic acid, 2 mM K2ATP, pH 7.4. The pipette was aimed at layers II–IV of the visual cortex and the stimulating electrode was placed in the white matter under the guidance of a stereomicroscope (Olympus Corporation, Tokyo, Japan).

Whole-cell recording was achieved using the patch-clamp technique[13, 33-34] with an Axoclamp-200B amplifier (Axon Instrument, Foster City, CA, USA). Postsynaptic currents were evoked with a stimulus of 0.5 mA and 100 μs duration, and the voltages were clamped at –30 mV. Series resistance (10 to 20 megohms) was compensated and checked for constancy throughout the experiments.

Bicuculline methiodide (20 μM)(Sigma, St. Louis, MO, USA) and D,L-2-amino-5-phosphonovalerate (20 μM) (Sigma) were added to artificial cerebrospinal fluid for at least 5 minutes, sufficient to block gamma- aminobutyric acid receptors and N-methyl-D-aspartate receptors, and the AMPA-EPSCs were recorded. The AMPA-EPSCs were also assessed in the presence of bicuculline methiodide (20 μM), 6-cyano-7- nitroquinoxaline-2,3-dione (20 μM)(Sigma) and D,L-2- amino-5-phosphonovalerate (20 μM) to block gamma-aminobutyric acid, AMPA and N-methyl-D- aspartate receptors, respectively. Finally, all blockers were washed out to test the recovery of postsynaptic currents[28].

讨论

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延伸阅读

1研究背景

弱视患者占世界人口的2-5%，其矫正视力低于正常，缺乏适应高科技发展所必须的高级视功能。中国有1000多万儿童弱视患者，是儿童低视力和致盲的主要原因之一。弱视是由于生后早期，异常的视觉环境如形觉剥夺、斜视、屈光参差和高度双眼屈光不正等导致的视觉发育异常。目前尚无治疗弱视的药物，仅采用遮盖等物理方法治疗，4-6岁时疗效好，但不能配合物理治疗；大于7岁时大多能够配合物理治疗，然而，7岁以后视皮质的可塑性基本被“抑制”了，异常视觉环境造成的弱视将难以治疗，治愈率不足18%[1]。如何重新激活视皮质被“抑制”的可塑性？是解决问题的关键，是儿科学、眼科学和神经科学的交叉领域，是亟待解决的临床难题。从分子机制研究着手，在细胞和突触水平上研究视皮质可塑性变化的机制，有助于寻找重新“激活”视细胞功能的途径，进而开发治疗弱视的药物和提高弱视治愈率。

人类和哺乳动物出生后，视觉系统能够根据视觉环境调整和改变与生具有的神经联系和突触结构。在视觉发育过程中，弱视的视功能障碍是由异常视觉环境造成，其主要改变发生在视皮质[2]。因此，近年来对弱视发病机理的研究主要是以视皮质可塑性作为突破口，包括细胞外和细胞内两方面的机制。

细胞外基质分子与视皮质可塑性的关系已引起关注[3-6]。硫酸软骨素是一种糖蛋白，密集于神经元周围形成的网状结构——神经元周围网络上，是中枢神经系统细胞外基质的主要成分。构成细胞外基质的神经元周围网络主要由硫酸软骨素、透明质酸和糖蛋白组成，完全包纳了神经元胞体和树突，围绕突触分布，在突触连接处成“孔”，主要作用是形成细胞外屏障[3]。在正常视觉环境下，生后22d大鼠视皮质硫酸软骨素分散分布，几乎没有神经元被神经元周围网络包绕，在生后35d神经元周围网络数目显著增高，至生后70d达到成年水平；在斜视的异常视觉环境下，猫视皮质硫酸软骨素阳性细胞数比正常猫减少。因此，认为硫酸软骨素参与神经元周围网络发育性成熟，导致视皮质可塑性的逐渐降低，标志着中枢神经系统突触回路的成熟[4-5]。采用软骨素酶ABC在体注入成年动物视皮质，降解硫酸软骨素可以恢复成年视皮质眼优势柱的可塑性[6]，提示视皮质眼优势柱可塑性的再“激活”与硫酸软骨素有关。然而，硫酸软骨素在视皮质可塑性中发挥作用的细胞内机制尚不清楚。

在弱视视皮质可塑性的细胞内机制研究中，谷氨酸受体的作用已受到重视[7-11]。已经证实：视皮质神经元的兴奋性突触释放谷氨酸，主要作用于2类离子型谷氨酸受体：α-氨基-3-羟基-5-甲基-4-异恶唑丙酸和N-甲基-D-天冬氨酸受体，产生突触后膜的去极化[7]。本实验室阴正勤等[8]在斜视性弱视猫上发现，N-甲基-D-天冬氨酸-R1 mRNA 构型与斜视性弱视猫视皮质神经元 N-甲基-D-天冬氨酸R1受体水平表达减少有关，证实 N-甲基-D-天冬氨酸参与了弱视视功能损害过程，且与视觉发育可塑性相关。之后，申请者等（2003）研究发现，大鼠视皮质NR1的表达呈年龄依赖性的过程，N-甲基-D-天冬氨酸受体介导的兴奋性突触传递功能变化的分子基础是基于 N-甲基-D-天冬氨酸受体亚单位 NR-1以外的亚单位构成改变。Philpot等[9]研究发现 N-甲基-D-天冬氨酸受体亚单位 NR-2A和NR-2B与视皮质神经元突触传递的功能可塑性有关。在视觉剥夺引起的视皮质突触抑制的可塑性机制中，α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体具有重要作用[10]，其亚单位 GluRs1和GluRs2的变化参与了视皮质可塑性双向改变[11]。在 N-甲基-D-天冬氨酸与α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体的关系研究上，申请者等[7]采用脑片膜片钳细胞内记录技术结合神经药理学的方法，对视觉发育可塑性关键期内正常和双眼形觉剥夺性弱视大鼠视皮质神经元的谷氨酸电流成分进行分离，率先揭示了双眼形觉剥夺通过 N-甲基-D-天冬氨酸和α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体的相互作用影响了视皮质神经元兴奋性突触传递的功能可塑性，从突触传递功能上证实N-甲基-D-天冬氨酸和α-氨基-3-羟基-5-甲基-4-异恶唑丙酸的相互作用参与了弱视视功能损害过程。N-甲基-D-天冬氨酸受体相对于α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体作用减弱的过程，与发育晚期硫酸软骨素表达增多的过程基本一致。那么，硫酸软骨素是否通过谷氨酸受体介导的细胞内机制抑制成年视皮质可塑性的？硫酸软骨素是否参与了形觉剥夺对视皮质神经元兴奋性突触传递功能可塑性影响的过程？目前尚未见文献报道。

基于上述认识，假设视觉发育过程中，细胞外基质硫酸软骨素通过细胞膜上的谷氨酸受体，引起细胞内通讯和信号变化，进而介导视皮质可塑性。那么，需要进一步证实：①去除硫酸软骨素的作用后，谷氨酸受体介导的突触后电流特性及其分子基础是否发生变化？如何变化？②去除硫酸软骨素的作用后，双眼形觉剥夺对谷氨酸受体介导的突触后电流特性的影响是否发生变化？如何变化？

为此，课题从硫酸软骨素和谷氨酸受体的关系入手，借助脑片膜片钳全细胞记录技术、受体电流分离和分子杂交等方法，研究正常和形觉剥夺性弱视大鼠视皮质硫酸软骨素与神经元α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体和N-甲基-D-天冬氨酸受体介导的兴奋性突触传递功能特性的关系，与α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体和N-甲基-D-天冬氨酸受体相互作用的关系及其分子基础，在细胞和突触水平上阐明硫酸软骨素抑制视皮质眼优势柱可塑性的机制及其与谷氨酸离子通道的关系，为恢复视皮质被抑制的可塑性提供思路，为儿童弱视的治疗奠定基础。