Using Viral-Mediated Gene Transfer to Study Depressive- Like Behavior - Viral Vector Approaches in Neurobiology and Brain Diseases - page 164

cases, the transgenic receptor can be activated by an endogenous
signaling molecule such as a neurotransmitter; since neurotrans-
mitter release is presumably unaltered by the overexpression of
postsynaptic receptors, this means that transgenic receptor activa-
tion will be governed by the dynamics of endogenous neurotrans-
mitter release. It is also possible to determine whether viral
overexpression of a receptor in a specifi c brain region increases the
potency of an agonist administered systemically.
One recent technology includes designed receptors that are
exclusively activated by designer drugs (DREADDs; see [ 4 , 5 ]).
The benefi ts of this system are obvious—receptors can be directed
to specifi c regions or cells, and turned off or with agents that have
no off-target effects. This technique is discussed in detail in the
Ferguson and Neumaier chapter. One can also “knockdown”
expression of an endogenous protein using viral expression of
shRNA to reduce the accumulation of newly synthesized proteins;
the rate of disappearance of the targeted protein will depend in part
on its half-life. In some cases, it may be advantageous to introduce
a mutated form of a protein either into wild-type animals or knock-
out animals that lack the gene of interest. Gain of function experi-
ments involving reintroduction of a protein into a knockout animal
is a powerful strategy for establishing the necessity and suffi ciency
of a protein for a particular function. Since many proteins must
dimerize in order to function, another strategy to reduce the func-
tion of a specifi c protein is to express a dominant negative mutant
protein using a viral vector; this has been used to study the role of
CREB in drug reward mechanisms, for example [ 6 ]. However, it
must be kept in mind that it is possible that such a mutant may also
interfere with the function of other related proteins. Thus, viral
vector strategies offer not only regionally and temporally precise
manipulations of brain function but they also allow innovative
methods for identifying which protein candidates are responsible
for a particular behavioral observation within that region.
Even though viral-mediated gene transfer is well tolerated,
there are several important controls that should be considered. It
is possible that the surgical procedure or nonspecifi c features of a
viral vector may alter the function of a brain region; therefore,
nonsurgical or sham surgical controls should be included whenever
a new brain region is targeted or major shift in method is initiated.
After that, we usually use fl uorescent protein expression as a
primary control group. We modifi ed the amplicon-based HSV vec-
tor system to contain a separate transcriptional cassette containing
CMV promoter and green fl uorescent protein (GFP), while expres-
sion of the transgene of interest was controlled by the HSV pro-
moter [ 7 ]. In most cases, we have expressed an epitope-tagged
transgene, such as hemagglutinin-tagged 5-HT 1B receptors, which
we confi rmed did not alter the functionality of this serotonin recep-
tor [ 7 ]. Our usual control group has been GFP alone, expressed