Abstract
Extracellular gradients of secreted guidance factors are known to guide axon pathfinding and neuronal migration. These factors are likely to bind to cell surfaces or extracellular matrix, but whether and how they may act in bound gradients remains mostly unclear. In this study, we have developed a new technique for rapid production of stable microscopic gradients of substrate-bound proteins by covalent bonding of the proteins with an epoxy-coated glass substrate while they are diffusing in an agarose gel. Using this method, we found that bound gradients of netrin-1 and brain-derived neurotrophic factor (BDNF) can polarize the initiation and turning of axons in cultured hippocampal neurons. Furthermore, bound BDNF gradient caused attractive and repulsive polarizing response on gradients of low- and high-average density of BDNF, respectively. This novel bidirectional response to BDNF depended on the basal level of cAMP in the neuron. Finally, our data showed that the neuron's attractive response to bound BDNF gradient depended on the absolute difference rather than the relative difference in the BDNF density across the neuron, with a minimal effective difference of 1-2 BDNF molecule/mum(2) on the substrate surface. Thus, substrate-bound guidance factors are highly effective in polarizing axon initiation and growth, and the diffusive printing technique is useful for studying neuronal responses induced by bound protein gradients.