Lowenstein D H, Arsenault L
Department of Neurology, University of California at San Francisco 94143, USA.
Neuroscience. 1996 Oct;74(4):1197-208. doi: 10.1016/0306-4522(96)00226-6.
The molecular mechanisms that underlie dentate granule cell axon (i.e., mossy fiber) growth during development and following seizure-induced hippocampal injury remain unknown. Part of this process may involve specific factors that support dentate granule cells during differentiation, and molecular cues that allow the appropriate growth of mossy fiber axons toward their targets. To study this process, we developed an in vitro assay system to measure the activity of putative trophic, chemoattractant and chemorepulsive factors. Two-hundred-micrometer-thick transverse hippocampal sections were prepared from neonatal rats and microdissected to isolate the middle one-third of the superior blade of the dentate granule cell layer. These were embedded in a three-dimensional collagen matrix either alone or with microdissected regions of the CA2 pyramidal cell layer. Cultures were maintained in a defined medium and grown for two to three days in a standard culture environment. Results showed that numerous processes grew primarily from the hilar side of explants into the collagen matrix, often in excess of 500 microns in length. These were determined to be axons based on: (i) morphological criteria including size and presence of growth cones, (ii) synaptophysin and growth-associated protein-43 immunoreactivity, (iii) lack of glial fibrillary acidic protein immunoreactivity and (iv) contiguity of biocytin-filled processes with neuronal soma within the explant. Treatment of cultures with brain-derived neurotrophic factor caused a significant increase in axon number and length, and this effect was partially reversed by the addition of a trkB-immunoglobulin fusion protein that blocks the activity of brain-derived neurotrophic factor and neurotrophin-4/5. Basic fibroblast growth factor also caused a marked increase in axon number and length, and caused a migration of neuron-like cells out of the explant into the collagen. These results show that cultured dentate granule cell layer explants are capable of growing mossy fibers into a neutral collagen matrix, and the growth of axons can be modified by the addition of exogenous growth factors. Furthermore, since target tissue and point sources of purified factors can easily be co-cultured with the explants, this new system provides a direct means for testing the molecular cues that influence mossy fiber growth.
在发育过程中以及癫痫诱发的海马损伤后，齿状颗粒细胞轴突（即苔藓纤维）生长的分子机制仍然未知。这一过程的部分原因可能涉及在分化过程中支持齿状颗粒细胞的特定因子，以及允许苔藓纤维轴突向其靶标适当生长的分子信号。为了研究这一过程，我们开发了一种体外检测系统来测量假定的营养、化学吸引和化学排斥因子的活性。从新生大鼠制备200微米厚的海马横向切片，并进行显微切割以分离齿状颗粒细胞层上叶片中间的三分之一。将这些切片单独或与CA2锥体细胞层的显微切割区域一起包埋在三维胶原基质中。培养物在限定培养基中维持，并在标准培养环境中生长两到三天。结果表明，许多突起主要从外植体的门区一侧生长到胶原基质中，长度通常超过500微米。基于以下几点确定这些为轴突：（i）包括大小和生长锥存在的形态学标准，（ii）突触素和生长相关蛋白-43免疫反应性，（iii）缺乏胶质纤维酸性蛋白免疫反应性，以及（iv）生物素填充的突起与外植体内神经元胞体的连续性。用脑源性神经营养因子处理培养物导致轴突数量和长度显著增加，并且通过添加阻断脑源性神经营养因子和神经营养素-4/5活性的trkB-免疫球蛋白融合蛋白，这种作用部分逆转。碱性成纤维细胞生长因子也导致轴突数量和长度显著增加，并导致神经元样细胞从外植体迁移到胶原中。这些结果表明，培养的齿状颗粒细胞层外植体能够将苔藓纤维生长到中性胶原基质中，并且通过添加外源性生长因子可以改变轴突的生长。此外，由于靶组织和纯化因子的点源可以很容易地与外植体共培养，这个新系统为测试影响苔藓纤维生长的分子信号提供了一种直接方法。
