Viral Vector-Based Techniques for Optogenetic Modulation In Vivo - Viral Vector Approaches in Neurobiology and Brain Diseases - page 281

to assure that the desired effect is achieved in the targeted cells.
The extent of viral transduction depends both on the type of vector
used and the brain region targeted. Generally, a relatively restricted
expression pattern can be achieved by choosing the appropriate
viral vector and injection volume. For example, AAV2 injection
results in expression patterns that are more localized compared
with the pseudotyped AAV2/5, AAV2/8, or AAV2/9. AAV2 is
therefore well suited for local expression in volumes smaller than
1 mm 3 [ 18 ]. Although viral titer can be reduced in order to
decrease the size of the transduced volume, lower titer injections
are also likely to infl uence the number of genome copies in trans-
duced cells, leading to lower expression levels of the transgene in
individual cells within the region [ 18 ]. On par with AAV2 trans-
duction, LV transduction is more spatially restricted in vivo and
can thus be used to target smaller structures. However, LV has
been reported to exhibit a bias towards excitatory neurons in cor-
tex [ 23 ], an effect which is likely also region specifi c, since other
more specialized cell types have been successfully targeted with
lentiviral vectors [ 24 , 25 ].
If the target area is large, there are several possible limitations
to the spatial extent of tissue that can be affected through optoge-
netic techniques. In the case of optogenetic inhibition, light-gated
ion pumps such as NpHR [ 8 ] and Arch [ 9 ] are commonly used.
These opsins have been optimized for neuroscience applications by
adding various targeting motifs and are effi ciently expressed in neu-
rons [ 13 , 26 ]. Due to the need for continuous illumination for
activation of these ion pumps, the limiting factor in experiments
utilizing these tools is the light power density required to achieve
effective hyperpolarization of the expressing neurons. Since brain
tissue strongly absorbs visible light and is a highly scattering
medium, light power density drops to ~1 % within 1 mm from the
light source (although this effect is strongly wavelength dependent;
see references [ 9 , 10 , 24 , 27 ] for more details). The maximal appli-
cable light power for optogenetic modulation is further limited by
its cytotoxic effect above certain irradiance levels. A power density
of 100 mW/mm 2 is regarded as the upper bound for direct light
stimulation in brain tissue, thereby restricting the volume of tissue
that can be modulated with optogenetic tools requiring intense
light for effective modulation (for example, the effective power
dose required for eNpHR3.0 is 5 mW/mm 2 , while most channel-
rhodopsin variants require less than 1 mW/mm 2 [ 13 ]). A single
injection of 1 l of AAV2/5 or AAV2/1 can lead to expression in a

region >1 mm 3 (see Fig. 3 for a representative example). If the
opsin expressed is eArch3.0 or eNpHR3.0, the volume affected by
light stimulation through an implanted optic fi ber in this region is
likely to be smaller than the transduced volume [ 28 ]. Therefore, in
this scenario the transduction volume is normally not restrictive.
In the case of neuronal excitation, new tools such as the step-
function opsins (SFO [ 29 , 30 ]) can be used to effectively

μ