Actin Isoforms in Neuronal Development and Function - International Review of Cell and Molecular Biology - page 198

tremendous potential within areas yet to be explored, it is truly an exciting
time for the study of actin isoforms in neuronal development and function.
In this section, we will outline what we believe to be some of the critical
short- and long-term experiments that will be needed in order to advance
our understanding of the role of actin isoforms in neurons and the ner-
vous system. The experiments outlined here complement the more specific
experiments proposed in the preceding sections.
7.1. Molecular and Cellular Studies
Perhaps the most obvious and pressing question that remains to be answered
is whether β-actin is specifically required for growth cone turning.The most
direct way to answer this question is with an in vitro growth cone turn-
ing assay to eliminate potential confounding factors in more complicated
systems. Until recently, this type of experiment was only routinely possible
in cultured Xenopus neuronal systems. However, Welshhans et al. recently
demonstrated that growth cone turning assays could be reliably performed
using primary mouse cortical neurons in a neuron “ball” culture system
( Welshhans and Bassell, 2011 ). Utilizing the same experimental setup with
neurons cultured from a β-actin KO mouse ( Cheever et al., 2012 ) could
provide direct evidence as to whether β-actin is required at all for growth
cone turning in a mammalian system. Interpretation of a positive result is
relatively straightforward, but a negative result could also be due to genetic
reprogramming of β-actin null neurons. The use of a recently described
method to monitor spatiotemporal changes in G-actin concentration ( Kiu-
chi et al., 2011 ) is one technique that could be used to ascertain whether
transcriptional programs may be altered as seen in β-actin KO fibroblasts.
Cultures of β-actin null neurons could also be allowed to mature where
dendritic spine formation, dynamics, and function could be directly assessed.
In particular, miniature excitatory postsynaptic currents (mESPCs) could be
measured in cultures of β-actin null neurons and compared to controls in
order to gain insight on synaptic roles for β-actin. With the proper controls,
any reduction in the amplitude of the mESPCs in β-actin KO neurons
would suggest that dendritic spine and postsynaptic function is impaired.
Positive results could then be followed up by staining for neurotransmitter
receptors given that postsynaptic actin has been implicated in the regu-
lation of the surface levels and localization of these receptors. The same
experiments could also be performed with γ-actin null neurons in order
to determine whether there are any isoform-specific roles in the regulation
of dendritic spine function. These experiments promise to shed significant