Precocious axons and improved survival of rat hippocampal neurons on lysine–alanine polymer substrates

Abstract

We tested the hypothesis that other polymers of lysine would be better substrates for culture of CNS neurons than polylysine itself. In a serum-free medium optimized for survival of hippocampal neurons grown on substrates of poly- d-lysine, 13% more neurons survived on substrates to which a sequential copolymer of lysine and alanine (LAS) was applied ( P=0.006). The effect was specific for the sequential polymer, in contrast to the random copolymer of lysine and alanine. This suggests that average cationic charge density is not as important as the spacing of these charges. More dramatically, immunostaining for the axon-associated microtubule-associated protein, tau, indicated a 2-fold higher rate of fiber growth on LAS. The somatodendritic cytoskeletal component MAP2 also appeared to be increased in cells cultured on LAS. This suggests that cytoskeletal differentiation in general and axon formation in particular are stimulated by the LAS substrate. Scanning electron microscopy supported this conclusion. By circular dichroism, the conformation of LAS in phosphate-buffered saline appeared to be a random coil, indistinguishable from poly- d-lysine. These results indicate that LAS is a superior substrate to polylysine for growth of CNS neurons. LAS may be useful for regeneration of damaged circuits in the CNS as well as a substrate for connections to a neuroprosthesis.