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targeted virus-mediated expression by the use of the enkephalin
and dynorphin promoters.
Finally, the successful implementation of bacterial artifi cial
chromosome (BAC) or knock-in technologies lead to transgenes,
which exhibit direct opsins expression. Among them are mouse
lines expressing opsins under the Thy1 promoter. Thy1 drives ran-
dom expression in a subset of neurons, resulting in generation of
several distinct lines [
37
-
39
]. More restricted expression was
achieved using the cholinergic ChAT promoter: [
40
]; serotonergic
TPH2 promoter: [
40
]; GABAergic VGAT promoter: [
40
]; gluta-
matergic VgluT promoter: [
41
]; parvalbumin ergic Pvalb pro-
moter: [
40
], or for olfactory sensory neurons the OMP promoter
[
42
]. A very elegant transgenic approach for neuropeptide-focused
research was accomplished recently by Tsunematsu et al. [
43
], who
achieved the expression of enhanced
Natronomonas pharaonis
halorhodopsin (eNpHR; a light-activated chloride pump that leads
to neuron hyperpolarization upon “yellow light” (580 nm), illumi-
nation) in HCRT neurons (94 % specifi city) of the lateral hypo-
thalamus. Complementing the previously described HCRT neuron
studies with ChR2/light-dependent activation leading to increased
awaking probability [
19
,
20
], here light application to eNpHR
(~573 nm) inhibited HCRT neurons and induced slow-wave sleep
in transgenic mice [
43
].
2.2 Indirect Viral
Approach Taking
Advantage from
Cre- Recombinase
While virus-based targeting of neuropeptidergic (and other types
of) neurons is often unsuccessful, an alternative technique is design-
ing transgenic driver lines, which can fully capitalize on extensive,
nonconstricted promoter and modulatory regions to drive recombi-
nase expression at best possible specifi city. Although the generation
of transgenic mice is rather laborious, time-consuming and expen-
sive, driver lines have huge advatages. In combination with recom-
binase-dependent systems-favorable viral vectors (but also lines used
for crossbreeding), which meet the requirements of very low to
absent activity in recombinase-free space-after recombination a
strong but still highly specifi c driving force for opsin expression is
granted. Different modes for recombinase-dependent vectors have
been developed; the integration of an LSL (loxP-STOP-loxP)-cas-
sette between promoter and opsin gene prevents unintended expres-
sion [
44
]. However, due to size limitations in viral vectors, the
shortened STOP-cassette is prone to occasional “read-through”
events, leading to leakiness even in the absence of recombinase Cre
[
44
,
45
]; Grinevich, personal observations). A further approach
involves the usage of inverted opsin genes and was termed FLEX
switch [
46
] or DIO (
double
-
fl oxed inverted open reading frame
)
[
45
,
47
,
48
]. In this approach, the combination of two pairs of het-
erologous lox-sites around the coding region ensures the gene turns
back in legible, upright orientation only after Cre activity (overview
of vectors with inter alia DIO-composition at:
http://www.stan-
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