Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 128

Also, as control, it is essential to always include neurons only
infected with GFP (e.g., Fig. 5 ), to ensure that any phenotype
observed in not due to infection of the viral particles or GFP
expression per se but due to the expression of the protein of
interest.
We have found that 24-h post in vivo infection with sindbis
viruses is enough time to allow for strong expression of the recom-
binant protein and observe alterations of synaptic function (e.g.,
[ 14 ]). If this protocol is adapted for use of other types of viruses,
such as the lentiviruses or the AAV, longer times will be required
between in vivo infection and electrophysiological analysis (usually
between 1 and 2 weeks).

6 Conclusions

In this chapter, I have described the use of a viral-based in vivo
gene expression system to study the role of specifi c proteins in syn-
aptic function, using the sindbis virus system as an example. This
procedure can however be adapted to any viral system that is neu-
rotropic. I believe that this technique offers several advantages over
other approaches that are used to express recombinant proteins in
vivo. In particular, it allows evaluation of the effect of expression of
any protein of interest in a temporally and spatially restricted man-
ner while minimizing the possibility of time-dependent compensa-
tions in response to the molecular manipulation. It also permits
direct comparison of molecularly manipulated and neighboring
control neurons within the same tissue under close-to-ideal physi-
ological conditions for analysis of synaptic function. One drawback
of the use of Sindbis viruses, however, is that they often result in
high over-expression of the recombinant protein. This, in theory,
could affect the normal functioning of the neuron as well as result
in the recombinant protein having effects that the endogenous
protein does not. Thus, I encourage the reader to keep up to date
with the latest versions of the Sindbis viruses that show the least
cytotoxicity and to consider the use of other viral-expression sys-
tems, such as lentiviruses or AAV, for longer-term expression
studies.

Acknowledgments

I thank Franck Aguila for design of fi gures and Fabien Lanté for
comments on the manuscript.