Viral Vectors for Optogenetics of Hypothalamic Neuropeptides - Viral Vector Approaches in Neurobiology and Brain Diseases - page 302

short promoters of OT and vasopressin (VP) genes fi rst in trans-
genic mice [ 9 ] and later by rAAV [ 10 , 11 ]. In previous and recent
works, Gainer's group demonstrated that an appropriate expres-
sion in magnocellular OT and vasopressin (VP) neurons can be
achieved with 5 gene-adjacent sequences of about 600 bp for OT

and 2 kb for VP, and eventually even 200-300 bp for each gene
promoter [ 10 , 11 ].
Another approach to uncover eligible OT and VP promoter
regions is to screen for conserved sequences that surround the
gene using the alignment software BLAT of the University of
California, Santa Cruz ( http://genome.ucsc.edu/cgi-bin/hgBLAT )
[ 8 , 12 , 13 ]. This can reveal functional and specifi city-defi ning
sequences of the promoter for this gene. The identifi ed mouse pro-
moter sequences of 2.6-kb length for OT and 1.9-kb length for VP
allowed for cell-type-specifi c expression in both magno- and par-
vocellular hypothalamic neurons [ 8 , 12 , 13 ]. Furthermore, these
short promoters preserved responsiveness to physiological stimuli
such as lactation or osmotic challenge. Most notably, cell-type
specifi city was accomplished without employing inter- and intra-
genic regulatory regions (as used by Gainer's group), successfully
meeting the size requirement for application in rAAV that has a
capacity limit of 5 kb. Sequence conservations were determined
from genome alignments of mouse and eight further mammalian
species and suggested a broad promoter specifi city and functional-
ity not only in rodent species, such as mice, rats [ 8 ], and voles
[ 13 ], but also in other mammals including (non-human) primates
and humans.
Importantly, expression levels of ChR2-mCherry (driven by
rAAV equipped with 2.6-kb OT promoter) in hypothalamic-OT
fi bers descending into the lateral part of the central amygdala
(CeL) were suffi cient for light-evoked OT release in the CeL as
measured by reduction of freezing time in contextual fear-
conditioned rats [ 8 , 14 , 15 ].
Correlation between the results from both approaches for pro-
moter design suggests that the shortened promoter (at least, in
case of the OT gene) is suffi cient for selective expression. However,
two recent studies, which used relatively similar sizes of OT pro-
moters (~ 600 nb), introduced either in rAAV [ 16 ] or in lentivirus
[ 17 ], lead to confl icting results. Both groups accomplished an
expression specifi city of >90 % in OT neurons. Atasoy et al. [ 16 ]
successfully aimed to activate parvocellular OT neurons by stimu-
lating cell bodies in the PVN, thereby overwriting AGRP ( agouti -
related protein ) neuron-mediated OT cell suppression and
modulating feeding behavior. Pinol et al. [ 17 ] did not achieve
functional stimulation of OT parvocellular-descending axons in
brain stem autonomic regions. Irrespective of whether lower OT
expression in parvocellular vs. magnocellular neurons or the usage
of minimal promoters account for defi cient axonal endowment

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