Viral Vectors for Optogenetics of Hypothalamic Neuropeptides - Viral Vector Approaches in Neurobiology and Brain Diseases - page 301

the seaweed Chlamydomonas reinhardtii ; after stimulation with
473 nm light (i.e., “blue light”), it rapidly opens a cation perme-
able channel, which depolarizes the cell and initiates action poten-
tials under precise temporal control [ 6 ].
To date, viral vectors represent the most popular means to
deliver optogenetic tools to neural systems. Most advantageous is
the rapidity of the experimental implementation and the ability of
temporally and spatially defi ned intervention. The recombinant
adeno-associated viral (rAAVs) vectors are especially useful, as they
display an extremely low immunogenicity. This increases their
potential to drive adequate expression for photocurrent stimula-
tion due to multiplicity of infection. In addition, they exhibit high
viral titers (usually ~10 13 genomic copies per ml; [ 7 ]). Furthermore,
rAAV is one of the smallest DNA viruses (20 nm diameter; [ 7 ]),
which allows effi cient spread in extracellular spaces to cover brain
regions of about 1 mm 3 (with 300-nl injection volumes; Knobloch
and Grinevich, personal observation). As a result, rAAV is able to
infect a complete brain nucleus such as the hypothalamic PVN or
SON (paraventricular or supraoptic nucleus), and render all neu-
rons of one given cell-type light sensitive [ 8 ]. Specifi city of expres-
sion can be achieved in two ways (1) either directly by employing
a selected gene promoter that drives viral expression of microbial
opsins in a certain cell population of similar phenotype or (2) indi-
rectly by delivering recombinase-dependent viruses into trans-
genic animals, which carry Cre (or other type) recombinase in
cell-type-specifi c manner, hence ensuring a selective viral opsin
expression. In the following text, we highlight both approaches,
discuss their limitations and advantages, and illustrate the fi ndings
with examples.

2 Material and Methods

2.1 Direct Viral
Approach and Specifi c
Promoter Design

Neuropeptidergic cells are located in deep, subcortical brain
regions, such as the hypothalamus, and often occur in intermin-
gled assemblies. Direct genetic access to distinct neuropeptidergic
cell types is diffi cult to accomplish because the regulatory elements
and mechanisms for specifi c expression may differ in between simi-
lar cell types, located in different brain regions and even in between
parvo- to magnocellular variants of the same cell type, located
within the same structures (such as the PVN). However, for a
small number of neuropeptides, the promoter design has resulted
in successful, cell-type-specifi c intervention. Among neuropep-
tides, most viral and genetic studies involved the use of oxytocin
(OT) neuron-specifi c promoters. The search for cell-type-specifi c
expression in OT and VP neurons is an “experimental odyssey” of
Harold Gainer, his coworkers and colleagues, who tackled genetic
targeting of magnocellular hypothalamic neurons by employing