The Journal of Neuroscience, March 15, 2000, 20(6):2266-2274
Growth Cones Are Not Required for Initial Establishment of
Polarity or Differential Axon Branch Growth in Cultured Hippocampal
Neurons
Gordon
Ruthel and
Peter J.
Hollenbeck
Department of Biological Sciences, Purdue University, West
Lafayette, Indiana 47907
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 undifferentiated minor neurites preferentially grows
to become the axon. Once the axon has formed, it typically branches,
and the branches grow differentially rather than concurrently. In axons
with only two branches, growth alternates between branches. In both
axon establishment and branch growth alternation, growth among sibling
processes or branches must be differentially regulated. We found that
elaborate and dynamic growth cones were associated with growth, whereas
diminished growth cones were associated with nongrowing processes or
branches. To test whether growth cones were necessary for differential
growth, growth cone motility was eliminated by application of
cytochalasin E. Although cytochalasin treatment before axon formation
yielded longer processes overall, a similar percentage of both treated
and untreated neurons had one process that grew more rapidly and became
much longer than its sibling processes. Immunostaining to visualize
dephospho-tau, an axonal marker, demonstrated that these single
dominant processes were axons. Axons that formed in cytochalasin were
thicker and showed more intense anti-tubulin staining than their
sibling processes. Branched axons deprived of growth cones retained a
pattern of differential growth and often included alternation. These
results indicate that neither formation of a single axon nor
differential growth of branches are dependent on growth cone motility
and suggest that the neuron can regulate neurite elongation at sites
other than at the growth cone.
Key words:
growth cone; axon outgrowth; cytochalasin; polarity; cell
culture; sibling bias
Copyright © 2000 Society for Neuroscience 0270-6474/00/2062266-09$05.00/0
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