Biomedical Engineering Reference
In-Depth Information
rhodopsins are members of the GPCR family, it is theoretically possible to design
synthetic rhodopsin-GPCR chimeras that combine the light-responsive elements of
rhodopsin with the machinery of biochemical signaling of specifi c GPCRs (Kramer
et al.
2009
; Karnik et al.
2003
; Kim et al.
2005
).
Optogenetic Gene Delivery Strategies
The commonest method of delivering optogenetic transgenes into the nervous sys-
tem is to infect cells with a self-inactivating virus, typically a lentivirus or adeno-
associated virus (AAV), that contains the transgene of interest driven by a short
promoter or enhancer element (Luo et al.
2008
). Cell-type-specifi c promoters that
have been used to drive optogenetic transgenes include EF1a (strong, ubiquitous
expression) (Tsai et al.
2009
; Cardin et al.
2009
), CAMKII-
(expression limited to
excitatory neurons) (Boyden et al.
2005
; Gradinaru et al.
2009
), synapsin I (limited
to neurons) (Zhang et al.
2007a
,
b
), and GFAP (limited to astrocytes) (Gradinaru
et al.
2009
). Several other promoters are useful for targeting to specifi c cell types in
the brain, such as the ppHcrt promoter that targets hypocretin-expressing neurons in
the lateral hypothalamus (Adamantidis et al.
2007
), the oxytocin promoter that tar-
gets oxytocin peptide hormone-releasing neurons of the hypothalamus (Knobloch
et al. 2012), and the synthetic PRSx8 promoter, which targets noradrenergic and
adrenergic neurons that express dopamine beta hydroxylase (Abbott et al.
2009a
;
Abbott et al.
2009b
). In utero electroporation is also useful for introducing optoge-
netic transgenes at specifi c developmental stages. Transgenes can be delivered to
specifi c cortical layers of the brain by electroporating mice at embryonic day E12.5
(layers V and VI), E13.5 (layer IV), or E15.5 (layers II and III) (Zhang et al.
2010
).
Several studies have used this approach to deliver ChR2 to specifi c cortical layers
for subsequent photostimulation when the mice reach adulthood (Gradinaru et al.
2007
; Hull et al.
2009
; Adesnik and Scanziani
2010
).
Viral, transgenic, and in utero electroporation strategies can be combined to
overcome the weak transcriptional activity of most endogenous promoters (Zhang
et al.
2010
). AAV vectors expressing Cre-dependent transgene cassettes under the
control of strong, ubiquitous promoters such as EF1a have been developed which
allow us to utilize numerous transgenic mouse lines from individual labs, such as
GENSAT (Gong et al.
2007
) and the Allen Institute for Brain Science (Madisen et al.
2010
), that express Cre recombinase in specifi c cell types. Indeed, many optoge-
netic studies have capitalized on these excellent systems (Tsai et al.
2009
; Cardin
et al.
2009
). Additionally, it is possible to specify the expression of optogenetic
transgenes using anatomical-based cell targeting (Gradinaru et al.
2010
). Rabies
viral vectors or proteins such as wheat germ agglutinin or tetanus toxin fragment C
help the gene of interest or Cre recombinase to be transported anterogradely and
retrogradely (Luo et al.
2008
; Gradinaru et al.
2010
; Zhang et al.
2010
; Osakada
et al.
2011
; Wall et al.
2010
). Thus, it is possible to restrict the expression of opto-
genetic transgenes in a particular neural circuit even if its cells do not express unique
genetic regulatory elements. Finally, cellular indicators of functional activity,
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