斜视性弱视猫发育过程中视皮层神经元NMDA-R1表达的免疫组织化学电镜观察

目的　研究不同发育阶段斜视猫视皮层神经元N 甲基 D 天门冬氨酸受体亚基 1(N methyl D aspartatereceptorsubunit 1,NMDA R1)在超微结构水平的表达与变化。方法　幼猫 11只 ,其中 6只猫在 2周龄行 1只眼外直肌断腱术产生单眼内斜。依动物处死时的年龄分为 3组 :3周龄组(斜视手术后 1周 ) ,2只正常和 2只斜视幼猫 ;5周龄组 (斜视手术后 3周 ) ,2只正常和 2只斜视幼猫 ;成年组 (6月龄 ) ,1只正常和 2只斜视性弱视猫。 3组动物均取初级视皮层组织进行冰冻切片 ,NMDA R1单克隆抗体标记后 ,分Ⅱ～Ⅲ层、Ⅳ层及Ⅴ～Ⅵ层各为一个区块行常规电镜染色切片 ,透射电镜观察。结果　 (1)电镜下分析 32 8个视皮层神经元 ,发现 3组正常猫的视皮层神经元NMDA R1标记阳性细胞数均高于斜视猫 (χ2 =4 2 8,4 4 1,4 89;P <0 0 5 )。 (2 )共计数 132 0个NMDA R1阳性突触 ,显示正常猫发育过程中 ,视皮层Ⅱ、Ⅲ层神经元细胞膜上的NMDA R1受体突触数多于Ⅳ～Ⅵ层 ,且随年龄增长而增加 (F =3 2 8,P <0 0 5 ) ;斜视猫视皮层神经元细胞膜上的NMDA R1受体突触数 ,3周龄组与正常幼猫组差异无显著意义 (F =0 17,P >0 0 5 ) ,5周龄组和成年组均较正常猫组显著减少 (F =2 6 94 ,4 7 0 1;P <0 0 0 1)。结论　 (1)正常猫发育过程

Electron microscopic analysis of expression of NMDA-R1 in the developmental process of visual cortex in strabismic amblyopic cat

OBJECTIVE: To investigate the expression and distribution of N-methyl-D-aspartate receptor subunit 1 (NMDA-R1) in neuronal ultrastructure in visual cortex of strabismic amblyopic cat during development. METHODS: Eleven kittens were used for this study. Esotropia in six kittens had been made monocularly by tenotomy at two weeks of age. Two pairs of normal and strabismic kittens were sacrificed in three weeks of age, one week after tenotomy. Another two pairs of normal and strabismic kittens were sacrificed in five weeks of age, three weeks after tenotomy. One normal and two strabismic amblyopic cats were sacrificed after 6 months of age. Animals were deeply anaesthetized and perfused transcardially with 4% paraformaldehyde. Cryostat sections of frontal central area P5-P0 were cut to 25 micro m thickness. The mouse anti-NMDA-R1 monoclonal antibody (mAb54.1, PharMingen) was used. After stained, a light microscope was used to select regions of layer II-III, layer IV and layer V-VI of visual cortex area 17 for re-embedding. HITACHI H-7000 transmission electron microscope at magnifications ranging from 30 000 - 300 000 X was used for observation. RESULTS: Three hundred and twenty-eight neurons of strait cortex were observed. NMDA-R1 receptor was located at the nuclei, Nissl body, cytoplasm, plasma membrane and the postsynaptic element of axons and dendrites. The ultrastructural morphology, including the mitochondrion, smooth endoplasmic reticulum, rough endoplasmic reticulum, and the Golgi apparatus, was not significantly different in the comparison between the cells in visual cortex of normal and strabismic groups. In the entire normal group, the percentage density of NMDA-R1 labeled cells was higher than that of strabismic groups (chi(2) = 4.280, 4.41, 4.89; P < 0.05). One thousand and three hundred and twenty NMDA-R1 immunopositive synapses were counted. The NMDA-R1 immunopositive synapses were dominated in layer II-III of visual cortex and increased during the development of normal kittens (F = 3.28, P < 0.05). There was no significant difference of NMDA-R1 immunopositive synapse distribution between the normal and strabismic kitten at 3 weeks (one week after operation) of age (F = 0.17, P > 0.05). The reduction of NMDA-R1 immunopositive synapse of plasma membrane in visual cortex of strabismic kitten was started at 5 weeks (threes weeks after surgery) of age. It was decreased significantly in strabismic amblyopic cat compared with that of the normal cat (F = 26.94, 47.01; P < 0.001). The ratios of nuclear membrane invagination of cells in visual cortex of normal and strabismic cat were higher than those of normal and squint kittens (chi(2) = 36.24, P < 0.01), but the ratio was not significantly different between the normal and strabismic group. CONCLUSION: (1) In the normal developmental process of cat, the plasticity of the neuronal synapsis in II and III layer of visual cortex is relatively great. (2) In the strabismus amblyopia occurring in the plastic critical period of visual development, no pathological changes of neuronal organelle in the visual cortex are found, but there are changes at molecular level in the neuronal synapsis.