Growth cones are not required for initial establishment of polarity or differential axon branch growth in cultured hippocampal neurons

Hippocampal neurons developing in culture exhibit two types of differential , seemingly competitive, process outgrowth in the absence of external cues. During the initial acquisition of polarity, one of several equivalent undi fferentiated minor neurites preferentially grows to become the axon. Once t he axon has formed, it typically branches, and the branches grow differenti ally rather than concurrently. In axons with only two branches, growth alte rnates between branches. In both axon establishment and branch growth alter nation, growth among sibling processes or branches must be differentially r egulated. We found that elaborate and dynamic growth cones were associated with growth, whereas diminished growth cones were associated with nongrowin g processes or branches. To test whether growth cones were necessary for di fferential growth, growth cone motility was eliminated by application of cy tochalasin E. Although cytochalasin treatment before axon formation yielded longer processes overall, a similar percentage of both treated and untreat ed neurons had one process that grew more rapidly and became much longer th an its sibling processes. Immunostaining to visualize dephospho-tau, an axo nal marker, demonstrated that these single dominant processes were axons. A xons that formed in cytochalasin were thicker and showed more intense anti- tubulin staining than their sibling processes. Branched axons deprived of g rowth cones retained a pattern of differential growth and often included al ternation. These results indicate that neither formation of a single axon n or differential growth of branches are dependent on growth cone motility an d suggest that the neuron can regulate neurite elongation at sites other th an at the growth cone.