【作者】 范惠民；

【导师】 阴正勤；

【作者基本信息】 第三军医大学 ， 眼科学， 2002， 硕士

【摘要】 目的：本课题拟通过视皮层脑片膜片钳全细胞记录法，结合细胞内标记技术，观察不同发育阶段双眼形觉剥夺大鼠视皮层神经元兴奋性突触后电流(EPSCs)变化及突触传递长时程增强现象(LTP)，研究形觉剥夺对发育过程中视皮层神经元突触电生理学和形态学特性的影响，探索哺乳动物视觉发育可塑性的细胞水平机制，为临床相关视功能障碍的治疗提供新的思路。方法：健康Wistar大鼠61只，分为正常组和双眼形觉剥夺组：正常组中3周龄（20-22天龄）13只，4周龄（27-29天龄）9只，5周龄（34-36天龄）10只；双眼形觉剥夺组3周龄（20-22天龄）12只，4周龄（27-29天龄）9只，5周龄（34-36天龄）8只。形觉剥夺组于13天时缝合双眼。制作脑片后利用盲法寻找细胞，获得膜片钳全细胞记录。通过视皮层的双极刺激电极给予0.07Hz的突触前测试刺激，记录视皮层神经元的PSCs。然后给予1Hz的低频刺激60~90个，每个脉冲伴随突触后去极化至-20mV 以诱发LTP。电生理记录结束后对所记细胞进行免疫组化染色。结果：1、所记录的PSCs分为3种类型：无反应型、单突触反应型和多突触反应型。双眼形觉剥夺组无反应型PSCs比例（20％）显著高于正常组（3.17％）(P<0.01)；多突触反应型PSCs比例则相反，正常组（27.2％）显著高于双眼形觉剥夺组（7.94％）(P<0.01)。2、双眼形觉剥夺组与正常组间相比，输入阻抗显著增高，静息电位较为去极化，EPSCs峰值显著降低，且形态幼稚。3、形觉剥夺组LTP诱发率显著低于正常组(P<0.05)。<WP=7>双眼形觉剥夺组组4周龄、5周龄的LTP增值显著高于正常组同周龄时（P<0.05）。结论： 1、大鼠视皮层多突触反应型神经元随发育有逐渐升高的趋势，而双眼形觉剥夺后大鼠视皮层无反应型神经元较正常大鼠显著增多，提示静息突触的激活可能是视皮层神经网络回路构建的一个重要基础。2、视皮层神经元电生理学特性和形态学特性紧密相关。形觉剥夺后大鼠视皮层神经元形态学的幼稚状态与功能上的不成熟是一致的。3、双眼形觉剥夺后大鼠视皮层神经元LTP诱出率显著下降，提示形觉刺激是视皮层神经元突触正常发育的重要条件。4、双眼形觉剥夺在降低视皮层神经元LTP诱发率的同时，可引起LTP幅值的明显提高。提示双眼形觉剥夺可在一定程度上降低视皮层神经元的可塑性，但可以保持神经元对适宜刺激的反应能力。更多还原

【Abstract】 Purpose: To study electrophysiological properties of neurons in visual cortex of binocular deprivated rats during the development and explore the synaptic and cellular mechanism of experience-dependent plasticity in the visual cortex. The relationship between morphological and functional maturation of neurons in visual cortex was studied with the biocytin.Methods: The visual cortex slices were prepared from 61 Wistar rats of both genders, which were divided into two groups: control group and binocular pattern deprivation (BD) group. In the control group, 32 rats were recorded at 3, 4 and 5 postnatal weeks. 13 rats were sacrified at 3 weeks of age (20~22days); 9 rats were sacrified at 4 weeks of age (27~29 days); 10 rats were sacrified at 5 weeks of age (34~36 days). In the BD group, 29 rats were recorded. 12 rats were sacrified at 3 weeks of age(20~22days); 9 rats were sacrified at 4 weeks of age (27~29 days) and 8 rats were sacrified at 5 weeks of age (34~36 days). Both eyelids were sutured at 13 days postnatal in BD group before the eyes open. Blind patch-clamp whole cell recording were adopted. Pre-synaptic test stimulation was given at 0.07 Hz intervals through bipolar stimulating electrodes. EPSCs of layer Ⅱ~Ⅳ were recorded by recording electrodes filled with 0.5% biocytin. Long-term potentiation (LTP) was induced by low-frequency stimulation (LFS) at 1Hz for 60~90 sec, each pulse of the LFS paired with depolarization of post-synaptic neurons to -20 mV. The neurons<WP=5>were stained after recording for the purpose of morphological study.Results: 1. There were three types of PSCs recorded: silent synaptic response, monosynaptic response, and polysynaptic response. The percentage of silent response in BD group (20%) was significantly higher than that in the control group(3.17%) (P<0.01) , While the incidence of polysynaptic EPSCs in BD group(7.94%) was significantly lower than that in the control group(27.2%) (P<0.01). 2. Compared with those of control group , neurons in BD group has a increased input resistance, peak value of EPSCs, a more depolarized resting membrane potential (P<0.05), and the neurons were morphologically less mature. 3. The incidence of LTP induced in BD group was lower while the LTP magnitude significantly higher than that in control group. Conclusions: 1. The input resistance and the resting membrane potential are important to discriminate the mature state of the neurons.2.The incidence of polysynaptic EPSCs tends to increase gradually in control group, while there are much more silent synaptic responses in BD group. It suggests that the activation of silent synapse may relate to in formation of synaptic connections. 3. The electrophysiological and morphological properties of neurons are intimately correlated with each other. 3. The incidence of LTP induction was decreased in binocular deprivated rats suggests that the visual stimulation of patterns facilitate the maturing of the neurons in visual cortex. 5. The amplitude of LTP increased in BD group while the incidence of LTP induction was getting down compare with those of control one. It implys that the plasticity of neurons in visual cortex was decreased by BD, but the response ability to adequate stimulus still exist.更多还原