Lärkfors L, Lindsay R M, Alderson R F
Regeneron Pharmaceuticals, Tarrytown, New York, USA.
J Neurochem. 1996 Apr;66(4):1362-73. doi: 10.1046/j.1471-4159.1996.66041362.x.
The ability of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron-specific enolase-immunopositve cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, approximately 200%, following BDNF, NT-4/5 or NT-3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT-4/5 (46%), whereas NT-3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT-3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT-4/5, and NT-3, but not NGF, induced the rapid expression of the immediate early gene c-fos. Immunocytochemical double-labeling with antibodies to c-fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c-fos. After 4 days in vitro, both BDNF and NT-3 induced the formation of c-fos protein in calbindin-immunopositive neurons, whereas NT-4/5 did not. The latter results suggest that although BDNF and NT-4/5 have been shown to act through a common receptor, TrkB, it appears that the effects of BDNF and NT4/5 are not identical.
在取自胚胎第16天大鼠小脑的解离培养物中，检测了神经营养因子神经生长因子（NGF）、脑源性神经营养因子（BDNF）、神经营养因子-3（NT-3）和神经营养因子-4/5（NT-4/5）促进神经元存活和表型分化的能力。BDNF处理分别在培养8天和10天后使神经元特异性烯醇化酶免疫阳性细胞的存活率提高了250%和400%。这些神经元中的一个亚群，即通过钙结合蛋白染色鉴定的浦肯野细胞，在BDNF、NT-4/5或NT-3处理后，数量增加到大致相同的程度，约为200%。通过GABA免疫反应性鉴定的GABA能神经元数量，经BDNF处理后大幅增加（470%），经NT-4/5处理后适度增加（46%），而NT-3则无作用。NGF未能增加浦肯野细胞或GABA能神经元的数量。要在8天后使浦肯野细胞数量达到最大增加，需要在细胞接种后48小时内添加BDNF。相比之下，即使接种后96小时才进行处理，NT-3的反应也几乎相同。BDNF、NT-4/5和NT-3，但不是NGF，诱导即刻早期基因c-fos的快速表达。使用针对c-fos和钙结合蛋白的抗体进行免疫细胞化学双重标记，以鉴定通过诱导c-fos对神经营养因子处理产生反应的浦肯野细胞。在体外培养4天后，BDNF和NT-3均诱导钙结合蛋白免疫阳性神经元中c-fos蛋白的形成，而NT-4/5则没有。后一结果表明，尽管BDNF和NT-4/5已被证明通过共同受体TrkB发挥作用，但BDNF和NT4/5的作用似乎并不相同。
