Viral Vector-Based Techniques for Optogenetic Modulation In Vivo - Viral Vector Approaches in Neurobiology and Brain Diseases - page 284

techniques are the unidirectional spread, fast expression, and high
amplifi cation capabilities of the SAD B19 virus [ 58 ]. On the other
hand, the time course of survival of SAD B19-infected neurons is
limited with cell health diminishing about 2 weeks following infec-
tion [ 51 ], suggesting that for experiments requiring long-term
survival of transduced neurons, other systems might be required.
An additional method for trans-synaptic circuit tracing is the
use of Cre-tagged trans-cellular tracer proteins. Trans-cellular
tracer proteins can be transported across synapses after binding to
specifi c glycoconjugates on the membrane (reviewed in [ 59 ]).
When using these proteins to confer trans-synaptic transport func-
tion, it is possible to fuse proteins of interest to these tracers,
thereby allowing circuit tracing (with fl uorescent markers) as well
as delivery of a functional protein if desired (such as enzymes).
Taking advantage of the trans-cellular transport properties of wheat
germ agglutinin (WGA), ChR2 can be expressed in a subset of
synaptically connected neurons [ 26 ]. This system requires two
separate constructs and viral injection sites: the fi rst, a WGA-Cre
fusion protein for combined recombinase and trans-synaptic func-
tionality, which can be expressed using AAV and targeted stereo-
tactically to a defi ned brain region. Cre-dependent AAV vector,
conditionally expressing a microbial opsin gene, is then targeted to
a predicted trans-synaptic target region. Although this method has
been successfully demonstrated in cortico-cortical and hippocam-
pal circuits, the directionality of transport of WGA-Cre has not yet
been fully characterized and might well be circuit dependent. In
this type of experiment, prior knowledge of synaptic connections
between the targeted neuronal populations is necessary in order to
determine the secondary injection site. For a more unbiased means
of functional mapping, it is possible to introduce WGA-Cre into
transgenic mice that conditionally express fl uorescent reporter
proteins [ 60 ] or microbial opsins [ 57 ].
Although several retrograde trans-synaptic tracing approaches
have been described, anterograde-specifi c tracer viruses are not yet
suffi ciently optimized for optogenetic experiments. Currently, the
most promising viral tool for anterograde tracing is the H129
strain of herpes simplex virus (HSV) type 1 [ 61 ] which has recently
been developed for genetically defi ned, Cre-dependent, antero-
grade tracing [ 62 ]. A possible application of this technology would
be the insertion of Cre-dependent microbial opsins into the H129
genome enabling light-induced activation of downstream circuit
elements. A different strain of HSV1, which is taken up by axons
and transported retrogradely, has been used for expression of
ChR2 in neurons projecting to the site of injection. This allows for
photostimulation-assisted identifi cation of neuronal populations
(PINP [ 63 ]). Importantly, not all HSV viruses spread antero-
gradely, and the directionality depends on the specifi c strain used.
As with many other neurotropic viral vectors, the main