Viral Vector-Based Techniques for Optogenetic Modulation In Vivo - Viral Vector Approaches in Neurobiology and Brain Diseases - page 283

At the whole-brain level, optogenetic stimulation has also been
integrated with functional magnetic resonance imaging (ofMRI
[ 47 - 49 ]) for identifi cation of global circuits recruited by defi ned
local activity patterns. This method enables unbiased mapping of
functionally connected areas without a priori knowledge of circuit
connectivity and allows identifi cation of downstream loci.
Recently, several new viral techniques have been developed that
allow the expression of optogenetic tools in neuronal populations
that are defi ned by their pattern of synaptic connectivity. Integrating
these tracing methods with optogenetics allows combined anatom-
ical and functional dissection of defi ned circuits. Several recent
studies have described and optimized an elegant technique for trac-
ing monosynaptic retrograde connections with a modifi ed rabies
virus; this approach is based on a glycoprotein-deleted variant of
the rabies SAD B19 strain, SAD G [ 50 , 51 ]. The rabies virus gly-

coprotein (G), which is embedded in the viral membrane, is
required for trans-synaptic spread. By introducing the glycoprotein
gene in neurons prior to infection of the G-deleted mutant virus,
the virus spreads to presynaptic neurons and is restricted from fur-
ther spread due to the lack of this complementary glycoprotein in
newly infected neurons [ 52 ]. This enables the dissection of direct
connections originating from a population of defi ned neurons or
even from a single primary infected neuron [ 53 ].
Osakada et al. [ 54 ] incorporated ChR2 into the glycoprotein-
deleted rabies virus (yielding SAD

Δ

G-ChR2-mCherry) and suc-
cessfully displayed optical activation of presynaptic ChR2 expressing
cells. This system makes it possible to outline the function of spe-
cifi c connections through combination with electrophysiological
recordings or behavioral paradigms. For an additional layer of
specifi city, the primary viral transduction can be directed to geneti-
cally defi ned post-synaptic neuronal subtypes by using the avian
receptor TVA system [ 55 ]. To achieve specifi city, the SAD

Δ

G virus
is pseudotyped with an envelope protein from the avian sarcoma
and leukosis virus (ASLV). The TVA receptor, which is required
for infection by the pseudotyped rabies virus and is only found in
birds, is then expressed in the cells to be targeted for infection by
SAD

Δ

G along with the rabies glycoprotein. This allows the virus
to spread trans-synaptically exclusively from the TVA-expressing
cells to their presynaptic partners. Specifi city in retrograde trans-
port can also be achieved by targeting the rabies G protein gene to
specifi c neurons using the double-fl oxed Cre-based expression sys-
tem [ 56 ]. In this preparation, only Cre-expressing cells will harbor
the necessary machinery for retrograde transport of the recombi-
nant rabies virus. To broaden the microbial opsin repertoire avail-
able for monosynaptic retrograde tracing, one could introduce a
Cre recombinase-expressing SAD

Δ

G virus into transgenic mice
engineered for Cre-dependent ChR2/Arch/NpHR expression
[ 57 ]. The advantages of using rabies-based circuit tracing

Δ