Actin Isoforms in Neuronal Development and Function - International Review of Cell and Molecular Biology - page 179

brain is believed to be one of the primary guidance cues that guide axons
of the hippocampal commissure to the other side of the brain ( Barallobre
et al., 2000 ; Serafini et al., 1996 ; Steup et al., 2000 ).
Because the dorsal hippocampal commissure is also absent in CNS- Act-
b KO mice, we hypothesized that the axons of the corpus callosum lacked
their normal growth substrate and sought to determine if the hippocampal
neurons exhibited an impaired response to netrin-1. To accomplish this,
we took advantage of the previously described increase in hippocampal
neuron axonal branching following exposure to netrin-1 as a readout for
axonal responsiveness ( Popko et al., 2009 ). Axonal branching is relatively
simple to measure and shares many common features with growth cone
turning including localized actin polymerization and Ca 2+ signals ( Dent
et al., 2003 , 2004 ), although there are differences as well. Using this axonal
branching paradigm, we found that neurons devoid of β-actin protein by
immunofluorescence ( Cheever et al., 2012 ) exhibited a similar increase in
axonal branching to netrin-1 as compared to controls ( Fig. 4.3 ), suggesting
that downstream signaling from the netrin-1 receptor deleted in colorec-
tal cancer does not require local β-actin translation. Why β-actin-deficient
axons of the hippocampal commissure fail to cross the midline is still not
entirely clear, although we favor the hypothesis that disruptions in the local
hippocampal architecture (Section 5.1.2 ) likely play a prominent role since
only axons extending through this region appear perturbed. If β-actin was
Figure 4.3 Netrin-1 induced axonal branching in control and β-actin KO hippocampal
neurons. (A-B) Representative images of β III-tubulin-stained primary hippocampal neu-
rons cultured from control and CNS- Actb KO embryos. Neurons were treated with a bath
application of either PBS or 250 ng/ml netrin-1 for 3 days followed by fixation, stain-
ing and analysis. (C) β β-actin-deficient neurons showed a comparable increase in axonal
branching in response to netrin-1 as control neurons. * denotes significantly different
than control neurons treated with PBS. # indicates significantly different than β -actin KO
neurons treated with PBS. n > 100 neurons for each genotype from at least two inde-
pendent experiments. Scale bar 20 µm. Representative control images for comparison
from Cheever et al. (2012).