小鼠大脑新皮质片层化形成过程和细胞周期变化

目的 探讨小鼠大脑新皮质片层化的组织发生过程和细胞周期的关系,对有丝分裂后神经元在迁移中的细胞周期变化、神经细胞的增殖、神经元的迁移进行观察.方法 各日龄共计200只小鼠,应用免疫荧光法、5′-溴脱氧尿嘧啶核苷(BrdU)检测和DiI标记技术对胚胎期和出生后小鼠的大脑皮质进行形态学观察,对皮质BrdU和Cyclin D1阳性细胞密度进行测量.结果 皮质板最早在胚龄15d(E15)时形成,小鼠大脑新皮质深层(第Ⅵ～Ⅴ层)片层化进程开始于生后0 d(P0),皮质浅层(第Ⅳ～Ⅱ层)的片层化趋势开始于P5,P7时6层结构完全形成,但未呈现片层化特点,P14时小鼠大脑新皮质片层化完全形成,P30时片层化结构趋于稳定.在大脑新皮质片层化过程中,锥体细胞在E17时呈椭圆形,树突有小分支,在P15时发育成熟,呈锥形并有复杂的顶树突和基树突.BrdU检测发现,室管层和室管层下区有大量增殖的干细胞,在此期间由BrdU阳性细胞增殖生成的有丝分裂后神经元可以迁移到大脑新皮质;P0至P30,迁移到皮质板的有丝分裂后神经元逐渐减少.利用G1期特异性标记物Cyclin D1对有丝分裂后神经元的细胞周期进行分析发现,有丝分裂后神经元处于G1期,它们一旦定居到皮质板将退出细胞周期.新皮质中Cyclin D1阳性细胞数量呈抛物线变化,在P12达到峰值,P30后在皮质板只能发现少量的Cyclin D1阳性细胞.结论 小鼠大脑新皮质片层化过程经历了细胞增殖、分化与迁移,同时伴随着皮质板锥体细胞的成熟.神经细胞的增殖和迁移主要发生在胚胎期和生后早期,迁移的细胞主要处于G1期.有丝分裂后神经元的分化过程实际上是G1期到G0期的过渡,一旦在皮质板定居下来,有丝分裂后神经元将退出细胞周期,进入G0期.

Neocortical lamination and cell cycle in the mouse

Objective To explore the relationship between the neocortical lamination and cell cycle, especially the cell cycle alteration of postmitotic neurons in their migration, proliferation, and the cortical development. Methods BR>A total of 200 mice were used in this study. The immunofluorescent staining, DiI tracing and 5-bromodeoxyuridine (BrdU) assay were used to observe changes in the cerebral cortex of embryonic and postnatal mice. Densities of BrdU and Cyclin D1 positive cells of the cerebral cortex were measured. Results At postnatal day 0 (P0), the lamination of deep cortical layers (Ⅵ-Ⅴ) started. The trend of lamination of superficial cortical layers (Ⅳ-Ⅱ) began after P5. The six layers were fully formed at P7.At P14, the feature of lamination in the cerebral cortex was fully formed, and then stabilized at P30. In the process of lamination, pyramidal cells were oval-shaped and had small branches with their dendritics at embryonic day 17 (E17). At P15, the pyramidal cells were mature and appeared with complex apical dendrites and base dendrites. With BrdU assay, we found that there were many proliferative stem cells in ventricular zone and subventricular zone. In the meantime, the postmitotic neurons that were proliferated from BrdU positive progenitor cells migrated into parenchyma of the neocortex. Statistical analysis showed that migrating postmitotic neurons into cortical plate(CP) decreased gradually from P0 to P30.The cell cycle of the migrating neurons was analyzed with GSUB>1/SUB> phase specific marker, Cyclin D1.The study showed that migrating neurons were in GSUB>1/SUB> phase after mitotic division, and they exited cell cycle once settlement in CP. The number of Cyclin D1 positive cells in the neocortex changed with parabolic curve and peaked at P12. After P30, only a few cyclinD1 positive cells were found in CP. Conclusion The neuroproliferation, cell differentiation and neuron migration are involved in neocortical lamination, which company with the maturation of pyramidal cells in the cortical plate. The neuroproliferation and neuron migration mainly occur in embryonic and neonatal periods. The migrating postmitotic neurons are in GSUB>1 /SUB>phase. The differentiation of migrating postmitotic neurons is the transition from GSUB>1 /SUB>to GSUB>0/SUB>.Once they settle in CP, the postmitotic n