Studies on axonal guidance of retinal ganglion cells in three-dimensional culture

The optic axons of the adult retina originate from the retinal ganglion cells (RGC). These axons reach their targets by interactions of their growth cones with the optic pathway microenvironment. In the chicken embryo, the optic nerve fibers undergo decussation and cross over at the optic chiasm. Many different molecules play a role in this axonal guidance, both as attractants and repellents, possibly for different sets of axons. Recent work in our laboratory has demonstrated high levels of cell surface alkaline phosphatase (AP) around the chick optic chiasm. This ectoenzyme will necessarily convert some of the extracellular ATP (adenosine triphosphate) present in brain tissue into adenosine, a nucleoside for which optic axons are known to contain receptors and which may promote neurite extension. Depending on concentrations and conversion rates, chiasmatic AP might establish an adenosine gradient, with its high point at the chiasm for optic axon guidance. To test this hypothesis we developed an in vitro system to study the role of adenosine in optic axonal guidance during early retinal development. Using a 3-dimensional collagen-based system to culture retinal explants from 5-7 day chick embryos (the period when the optic axons navigate toward the chiasm), we visualized the growth of optic nerve fibers in response to various soluble molecules. Adenosine and related molecules were released from Elvax (ethylene vinyl acetate) resin implanted into the collagen containing the retinal tissue. Adenosine stimulated growth of long, discrete axons, whereas AMP (adenosine monophosphate) and guanosine, at most, marginally affected axon outgrowth. We also carried out experiments in which retinal explants and chiasmatic tissue were co-cultured in collagen matrices containing ATP. When explants from 5-day embryos were used in these co-cultures retinal axons extended preferentially toward the chiasm. Similar experiments using CTP (cytosine triphosphate) did not show this effect. We also observed decreased neurite outgrowth and decreased neurite length in cultures treated with both adenosine and an adenosine A2 receptor inhibitor -- DMPX (3,7-dimethyl-1-(2-propynyl) xanthine) compared to cultures treated with adenosine alone, supporting a role of adenosine in neurite outgrowth from retinal explants in 3-dimensional collagen cultures. Our results indicate that adenosine promotes outgrowth of retinal nerve fibers and these navigate toward high concentrations of soluble adenosine, and that chemoattractive adenosine gradients can potentially be generated by conversion of extracellular ATP to adenosine by ecto-AP on the surfaces of chiasmatic cells.