Viral Vectors for Optogenetics of Hypothalamic Neuropeptides - Viral Vector Approaches in Neurobiology and Brain Diseases - page 303

with ChR2, or if furthermore the disparity of applied viruses causes
differences in the results, a high number of ChR2 at the stimula-
tion site is certainly necessary to ensure robust photocurrent
responses [ 1 , 17 , 18 ].
Hypothalamic neurons expressing hypocretin (HCRT, or
orexin) were recently targeted in a specifi c manner by Adamantidis
et al. [ 19 ] and Carter et al. [ 20 ] with a 3.1-kb mouse prepro-
hypocretin gene promoter. This resulted in 97 % expression speci-
fi city of ChR2. Light stimulation in HCRT neurons of mice was
suffi cient to increase the probability of an awaking event irrespec-
tive of the time of day and sleeping state. Also downstream located,
arousal-promoting areas were affected, mirroring the course of
endogenous cell activity and HCRT release [ 19 , 20 ].
To bypass cell-type-specifi c expression limitations, microbial
opsins can be expressed from “strong” general promoters, such as
CaMKII , CAG, Thy1, or synapsin, following a recombina-

tion event. Cell-type specifi city can then be indirectly assured with
selective recombinase availability as discussed below.
These “strong” promoters can also be used to selectively target
opsins directly to the neurons (excluding glia, if not a specifi c glial
promoter such as GFAP was employed). Specifi city can be reached
by precise stereotaxic delivery of virus volumes [ 21 - 27 ] or by stim-
ulating distinct and remotely located projection trees [ 17 , 28 , 29 ].
This approach can be applied in intact mice, rats, or macaques in
order to observe the behavioral impact of light-induced activation
(see also [ 1 ]).
Although some of the studies were conducted with viruses
other than rAAV (in particular, lentivirus), rAAV vectors have been
shown to be most effi cient in transducing neurons of the forebrain
[ 30 , 31 ]. They enable stable expression for at least 1 year even in
episomal state [ 7 ], providing a large variability in cell tropisms for
distinct infection of cell-types or tissues [ 32 ] and, due to their low
immunogenicity, allow for insights in virtually unaffected wild-type
animals as well as in animal models of disease. These advantages
make rAAV preferable over other viruses for assessing behavior in
optogenetically manipulated, awake animals.
For the sake of completeness, also viruses should be considered
that mediate specifi c expression in neuropeptidergic neurons, but
were not yet used in optogenetic studies. For example, Tan and
colleagues achieved 80 % expression specifi city with a mouse soma-
tostatin (mSst) promoter delivered via rAAV [ 33 ]. The same pro-
moter (mSst) was further examined by Nathanson et al. [ 34 ],
comparing its application with lentivirus and rAAV and extending
the study by including also fugu somatostatin (fSst) sequences in
the experimental approach to inject mice, rats, and monkeys.
Furthermore, van den Pol et al. [ 35 ] achieved specifi city with
application of recombinant virus to melanin-concentrating hor-
mone neurons of the hypothalamus and Ferguson et al [ 36 ]

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