DENTATE GRANULE CELL LAYER COLLAGEN EXPLANT CULTURES - SPONTANEOUS AXONAL GROWTH AND INDUCTION BY BRAIN-DERIVED NEUROTROPHIC FACTOR OR BASIC FIBROBLAST GROWTH-FACTOR

The molecular mechanisms that underlie dentate granule cell axon (i.e. , messy fiber) growth during development and following seizure-induced hippocampal injury remain unknown. Part of this process may involve s pecific factors that support dentate granule cells during differentiat ion, and molecular cues that allow the appropriate growth of messy fib er axons toward their targets. To study this process, we developed an in vitro assay system to measure the activity of putative trophic, che moattractant and chemorepulsive factors. Two-hundred-micrometer-thick transverse hippocampal sections wire prepared from neonatal rats and m icrodissected to isolate the middle one-third of the superior blade of the dentate granule cell layer. These were embedded in a three-dimens ional collagen matrix either alone or with microdissected regions of t he CA3 pyramidal cell layer. Cultures were maintained in a defined med ium 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 mu m in length. These were determined to be axons based on: (i) morphologi cal criteria including size and presence of growth cones, (ii) synapto physin and growth-associated protein-43 immunoreactivity, (iii) lack o f glial fibrillary acidic protein immunoreactivity and (iv) contiguity of biocytin-filled processes with neuronal soma within the explant. T reatment of cultures with brain-derived neurotrophic factor caused a s ignificant increase in axon number and length, and this effect was par tially reversed by the addition of a trkB-immunoglobulin fusion protei n that blocks the activity of brain-derived neurotrophic factor and ne urotrophin-4/5. Basic fibroblast growth factor also caused a marked in crease in axon number and length, and caused a migration of neuron-lik e cells out of the explant into the collagen. These results show that cultured dentate granule cell layer explants are capable of growing me ssy 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 me ans for testing the molecular cues that influence messy fiber growth. Copyright (C) 1996 IBRO. Published by Elsevier Science Ltd.