Viral Vector-Based Techniques for Optogenetic Modulation In Vivo - Viral Vector Approaches in Neurobiology and Brain Diseases - page 282

depolarize neurons at very low light power densities (up to 3 orders
of magnitude lower power density values than required for
stimulation with wild-type ChR2). The slow closing kinetics of
these channels allows for the accumulation of open channels over
long periods of time. This allows for neurons in large volumes of
brain tissue to be depolarized with relatively low light power densi-
ties, especially if millisecond precise control of neuronal activity is
not needed, or even undesired [ 30 ]. Here, viral transduction vol-
ume can become a limiting factor. The transduction effi ciency, titer
and binding affi nity to the cell surface of the injected viral vector as
well as its spread radius can be tailored to the desired outcome, as
detailed below.
Recent advances in AAV pseudotyping techniques have resulted
in strongly enhanced transduction effi ciency [ 31 ]. AAV2/9 has
been shown to possess far superior transduction effi ciency com-
pared to wild-type AAV2 ([ 32 , 33 ]; for review see [ 34 ]) and hence
results in larger transduction volumes [ 35 ]. The distance of viral
spread is dependent on multiple factors, including particle size and
abundance of the cognate receptor for cell entry. One method to
overcome the small range of diffusion-based injection protocols is
rapid injection, resulting in convection as shown in 2006 by
Raghavan and colleges [ 36 ]. This method, referred to as
convection-enhanced delivery (CED) seems promising, but
requires special injection needles that can prevent viral backfl ow
along the needle insertion tract. Another option is the co-
administration of agents aimed at increasing the diffusion of viral
particles. Intravenous mannitol injection [ 37 ] can be used to
decrease intracranial pressure, resulting in increased diffusion dis-
tance. A 2 l injection of AAV2/9, delivered using CED combined

with systemic mannitol administration allows for the effective
transduction of complete brain regions such as the hippocampus,
including contralateral structures [ 38 ]. In the case of AAV2, which
binds to heparan sulfate proteoglycans on the cell surface, co-
infusion of heparin was reported to increase viral spread [ 39 ].
Although these advanced methods have not been explored directly
for optogenetic experiments, we expect that they will be useful
when the targeting of larger volumes is desired.

μ

1.2 Circuit-Based
Expression of
Optogenetic Tools

Functional dissection of intact neural circuits is one of the most
widely used applications of optogenetic techniques.
Channelrhodopsin can itself serve as a tool for anterograde circuit
mapping [ 40 , 41 ]. Introduction of fl uorescently tagged channel-
rhodopsins into a specifi c neuronal population in a defi ned brain
region enables the visualization and subsequent photoactivation of
projecting axon terminals throughout the brain. Simultaneous
electrophysiological recording at the projection site allows the
identifi cation of specifi c postsynaptic components of the circuit
both in vivo and in the acute brain slice preparation [ 40 , 42 - 46 ].