Development of Cell-Type-Specific Viral Vectors to Tease Apart the Neural Circuitry that Contributes to Drug Addiction - Viral Vector Approaches in Neurobiology and Brain Diseases - page 156

needle is retracted too rapidly, the vector will be pulled up the
needle track. The surgical wound is closed using sterile sutures
and thread and the animals are placed in a recovery chamber
and monitored until awake. Because the onset and duration of
gene expression varies by promoter, pilot experiments must be
conducted in order to determine the time course of gene
expression for a given promoter.
3. Once transgene expression levels are stable, the animals are
ready to begin behavioral testing. If the selected behavioral
protocol requires extensive training prior to the testing phase,
this training may occur before the stereotaxic injection of the
viral vector. If the viral vectors express DREADD receptors
(Designer Receptors Exclusively Activated by Designer Drugs)
[ 7 ], then it will be necessary to activate the DREADD recep-
tors with CNO prior to each test session. Although dose and
route may vary with species and strain, ip injections of 1-3 mg/
kg CNO 15 min prior to testing is suffi cient to produce behav-
ioral effects in rats [ 9 , 19 ] and doses of 0.3 mg/kg CNO have
been shown to be effi cacious in mice [ 20 ]. Strategies for acti-
vating channelrhodopsin-2 and other light-activated receptors
are rapidly evolving and are outside the scope of this chapter
but have been recently reviewed [ 10 , 21 , 22 ]. Because
DREADD receptors and optogenetic viral vectors are driven
by ligands and light, respectively, it is possible to do within-
subject control experiments where the test phase occurs in the
presence or absence of receptor activation.
4. Upon completion of the behavioral experiment, animals
should be perfused and the brains processed for histological
verifi cation of transgene expression. One of the major advan-
tages of viral vector strategies is the opportunity to localize the
site of manipulation exactly by visualizing fl uorescent protein
directly or by detecting transgenes with immunohistochemis-
try. While fl uorescence microscopy can be used to directly and
conveniently visualize proteins such as GFP, we have noted
that many more infected cells will be seen using immunohisto-
chemistry, even for molecules like GFP. In vivo cell specifi city
of transgene expression can be confi rmed using markers of all
of the cell types in a given brain region together with labeling
of the tagged transgene or fl uorescent fusion protein. If the
promoter does indeed restrict transgene expression to a spe-
cifi c cell type, then transgene markers will only colocalize with
markers of the targeted cell type. In addition to using immu-
nohistochemistry to identify colocalization of transgene and
key cellular markers, we sometimes use retrograde tracer injec-
tions to identify specifi c cell types. For example, we confi rmed
the high fi delity of specifi c transgene expression in striatopal-
lidal and striatonigral neurons by utilizing retrograde labeling