THE EFFECTS OF GROWTH-FACTORS ON THE SURVIVAL AND DIFFERENTIATION OF CULTURED DENTATE GYRUS NEURONS

Dentate granule cells (DGCs) are the principal cell population of the hippocampal dentate gyrus, and granule cells provide the main excitati on to the hippocampus proper via their messy fibers axons. Although it is well established that granule cells express various growth factors and growth factor receptors, the functional effects of growth factors on the normal development and response to injury of granule cells are relatively unknown. To address this question, primary cultures enrich ed in DGCs were prepared by microdissecting hippocampal slices from ne onatal rats and growing dissociated cells in defined media with added nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotr ophin-3 (NT-3), neurotrophin-4/5 (NT4/5), ciliary neurotrophic factor, basic fibroblast growth factor (bFGF), or vehicle. The effects on cel l survival and morphology were quantified by studying neuron-specific enolase-immunostained cells at various time points, plating densities, host ages, and growth factor concentrations. BDNF or bFGF significant ly increased both neuronal survival and differentiation by 30-80% comp ared with control cultures. Maximal effects were observed at relativel y longer time points (5-12 d), with younger cells (postnatal day 3-5) and at lowest plating densities. Addition of a trkB-IgG fusion protein that blocks the activity of BDNF or NT4/5 inhibited the effects of BD NF and attenuated the differentiation of cells cultured at high platin g densities. Furthermore, treatment of cultures with the kinase inhibi tor K252b specifically blocked the effects of BDNF, suggesting involve ment of trkB (the high-affinity BDNF receptor) in BDNF-induced differe ntiation. These results show that growth properties of cultured neonat al DGCs are influenced by exogenously applied BDNF or bFGF in a time-, age-, and density-dependent manner. The effect of plating density sug gests an endogenous expression of growth factors in these culture cond itions, and this is mediated in part by endogenous BDNF acting via a t yrosine kinase receptor. Combined with previous work showing that vari ous growth factors and their receptors are expressed by DGCs, these fi ndings provide strong support for the hypothesis that BDNF and bFGF in fluence both the growth and development of DGCs in vivo.