Development and Aging of Neuronal Synapse

(A)脊椎细胞骨架分析我们用抗DREBRAIN单抗M2F6结合的微珠制备树突棘中的细胞骨架蛋白复合体。制备的样品除DREBRAIN本身外，还含有肌动蛋白、肌球蛋白I、肌球蛋白II、肌球蛋白V和明胶蛋白。此外，该样本中还有许多其他未鉴定的蛋白质；因此，我们利用该样本作为免疫原，制备了单抗。因此，我们提出了抗肌球蛋白抗体和抗CaMKII抗体。抗肌球蛋白抗体特异性识别非肌肉肌球蛋白II重链，除树突棘外，还存在于细胞体和树突干中。这表明，脊椎细胞骨架的特征是肌动蛋白结合蛋白，而不是肌球蛋白。(B)脊椎细胞骨架在发育和衰老过程中的变化。在发育中的大脑中，Drebrin E存在于迁移的神经元以及生长的轴突和树突中。在成人大脑中，Drebrin A定位于树突状…更多的冰刺。在这项研究中，我们研究了在发育过程中，Drebrin从细胞体中消失并在树突棘中丰富的时间。用日本MBL抗DREBRIN单抗对小鼠大脑和小脑进行免疫组织化学染色，结果表明，DREBRIN免疫反应阳性细胞在出生后0~10天出现在胞体和树突中，但随着出生后天数的增加，DREBRIN的表达总体呈下降趋势。而14日龄大鼠仅在神经束内观察到Drebrin免疫反应呈点状分布。胞体和树突内几乎未见Drebrin免疫反应。这些数据表明，Drebrin的亚细胞定位在出生后12天左右发生了同步变化，表明神经元细胞骨架的最终成熟可能发生在出生后12天。应用100μM谷氨酸可减弱树突棘的Drebrin免疫反应。转基因实验表明，神经元中过表达的Drebrin A导致了脊髓长度的延长。这些结果表明，DREBRIN从树突干分布到树突棘的发育变化可能与突触功能的成熟有关。较少

(A) Analysis of spine cytoskeletonsWe prepare the cytoskeletal protein complex in the dendritic spine using the bead bound to anti-drebrin monoclonal antibody M2F6. The prepared sample contained actin, myosin I, myosin II, myosin V and gelsolin in addition to drebrin itself. Further, there are many other unidentified proteins in that sample ; therefore, we raised monoclonal antibodies using this sample as an immunogen. As a consequence we raised anti-myosin antibody and anti-CaMKII antibody. The anti-myosin antibody specifically recognized non-muscle myosin II heavy chan, which is present in cell soma and dendritic shaft in addition to dendritic spine. This indicates that spine cytoskelton is characterized by actin binding proteins but not by myosins.(B) The change of spine cytoskeletons during development and aging.In the developing brain, drebrin E is observed in the migrating neurons and within the growing axons and dendrite. In the adult brain, drebrin A is localized in the dendrit … More ic spines. In this study we investigated when drebrin disappeared from cell soma and enriched in dendritic spines during development. Immunohistochemical staining of the cerebrum and cerebellum using anti-drebrin monoclonal antibody (MBL, Japan) demonstrated that drebrin immunoreactivity was observed in the cell soma and dendrites from postnatal 0 day to 10 day, although drebrin expression as a whole was decreased according to the postnatal days. However, in 14-day-old rat, drebrin immunoreactivity was only observed as dot-like pattern in neuropil. Drebrin immunoreactivity was hardly observed within the cell soma and dendrites. These data indicates that drebrin subcellular localization was changed synchronously around postnatal 12 days, indicating that the final maturation of neuronal cytoskeleton may occurred at postnatal 12 days. Application of 100 μM glutamate weakened drebrin immunoreactivity at dendritic spines. Transfection experiments demonstrated that overexpression of drebrin A in neuron resulted the elongation of the spine length. These data indicate that developmental changes in the distribution of drebrin from dendritic shafts into dendritic spines may be related to the maturation of synaptic function. Less