Localized gene expression of axon guidance molecules in neuronal co-cultures

Axonal growth cones are guided to their targets by contact-dependent mechanisms or by diffusible chemotropic factors. Axon guidance by these factors typically involves culturing neurons on an acellular substrate which may not represent the in vivo biological environment. We developed two novel in vitro methods to create patterned gene expression of guidance molecules in a physiologically-relevant cellular environment. In the Matrigel™ assay, a droplet of adenovirus-Matrigel™ suspension was placed on astrocytes grown in Matrigel™. The adenovirus diffused through the gel and transduced underlying astrocytes, creating a radial infection gradient within a localized area. In the second model, recombinant adenovirus was bound to an anti-hexon antibody adsorbed onto stripe patterns of nitrocellulose. Once the cells were added, only those contacting the adenovirus were infected. The outgrowth pattern of chick DRG neurons on NGF, semaphorin 3A and brevican were studied. As expected, results showed robust axonal growth toward NGF as opposed to either secreted Sema 3A or membrane bound brevican, however subtle differences in axonal growth responses were observed in comparison to those obtained with less physiologically-relevant methods. Novel to this technology, the location and area of molecule expression can be controlled and manipulated in an intricate cellular environment.