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Typical AAV vectors can be produced at much higher titers
than LV (typical yields of AAV reach 10 13 genome-containing
capsids per ml, whereas the same amount of starting material will
yield ~10 9 VSVG-pseudotyped lentivirus). In spite of its huge
potential in brain research, several properties of AAV make its pro-
duction as a recombinant viral vector a technical challenge for most
neuroscience laboratories. One such property is the inherent insta-
bility of the inverse tandem repeat sequences (ITR's) required for
correct demarcation of the genomic sequence inserted into the
capsid, as well as for effective reverse strand synthesis which is a rate
limiting step for effi cient gene expression from AAV. In addition,
in the absence of the full complement of AAV genes and a helper
virus (such as adenovirus) in the laboratory setting, recombinant
AAV is not exuded from the cells, thus requiring the lysis of the
producer cell culture during the purifi cation process. This requires
several technically challenging downstream purifi cation steps such
as purifi cation columns or ultracentrifugation [ 22 ]. Although both
LV and AAV have been successfully used in optogenetic experi-
ments, most neuroscience laboratories that do not possess molecu-
lar biology and tissue culture capabilities choose to purchase
AAV-based vectors from core facilities offering virus production
services where commonly used viruses are often kept in-stock as
injection-ready aliquots.
In optogenetic experiments, several critical factors should be
taken into account in order to assure that the desired physiological
effect is evoked by light stimulation (1) expression of the optoge-
netic actuator should be robust and allow modulation with moder-
ate light power in order to avoid phototoxicity; (2) expression
should be restricted to the desired neuronal population, with mini-
mal “genetic leak” to nontargeted cell populations; (3) the method
used to express the selected tool should be well tolerated and non-
toxic to cells over the entire duration of the experimental period
(and ideally well beyond this time); (4) in behavioral experiments,
the physiological effect of the manipulation performed should
always be validated in one of several electrophysiological recording
methods to assure that the desired effect is achieved and to cor-
rectly interpret behavioral results. For example, acute slice patch-
clamp recordings can be performed to verify expression and the
effi cacy of light stimulation; extracellular recordings can further
provide important validation of the effect of the optogenetic
manipulation in the intact circuit.
1.1 Target Volume
Considerations
Depending on the scientifi c question, the required spatial distribu-
tion of transduced cells can vary dramatically. This necessitates
adjustment of the viral delivery procedure and viral vector type to
allow for effi cient transduction in the target tissue. Furthermore,
the choice of optogenetic tool and illumination method is critical
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