89the simultaneous control and recording of activity in cultured cells (Wang et al.
2007 ), paving the way for in vivo studies using photo switchable tethers.
Ultimately, the true power of photoreversible tethers was realized when the
Isacoff lab introduced this technology in zebrafish (Szobota et al. 2007 ). Zebraf-
ish expressing an iGluR cysteine mutant in a tissue specific manner were bathed
in MAG. After illumination with a hand-held ultraviolet lamp, larvae expressing
the iGluR displayed abnormal swimming responses in response to stimuli. When
illuminated with visible light, the swimming response was completely restored.
Furthermore, this phenotype was specific to the tissue expression of LiGluR as
expression throughout the entire nervous system led to complete paralysis, but ex-
pression in the heart led to no phenotypic changes. Later, this group limited LiGluR
expression to a single spinal neuron type, Kolmer-Agduhr cells, to identify their
role in spontaneous locomotion during zebrafish development, a function previ-
ously unknown (Wyart et al. 2009 ). These studies demonstrated that it is possible
to use light to manipulate LiGLuR in vivo making it possible to dissect the role of
neural circuits in behavior.
In addition to probing channel function and neuronal circuits, the light-gated bio-
reactive tethers have been used to restore retinal function in blinding disease models
(Caporale et al. 2011 ). Previous studies showed that retinal expression of activat-
ing rhodopsins could restore light sensitivity to rodent models of blindess (Carter-
Dawson et al. 1978 ; Bi et al. 2006 ; Tomita et al. 2007 ; Lin et al. 2008 ; Zhang et al.
2009 ). Therefore, the Flannery group delivered the fusion protein AAV-LiGluR into
adult mice intravitreally (Caporale et al. 2011 ). Injection of MAG and subsequent
exposure to ultraviolet light elicited a pupillary response in WT mice expressing
AAV-LiGluR and restored the response in mutant mice.
Despite the multiple advantages the light-gated system offered, it required the
genes encoding cysteine mutants to be delivered to and expressed in target cells. To
transition this technology to endogenous channels, Kramer and colleagues gener-
ated derivatives of MAL-AZO-QA called PALs. Similar to the original molecule,
the quaternary ammonium was attached to one side of the azobenzene moiety but
instead of a maleimide as shown in Fig. 3a, the reagent contained an acrylamide,
chloroacetamide, or epoxide group (Fortin et al. 2008 ). In this derivation, selec-
tivity comes from the quaternary ammonium increasing the local concentration of
the electrophile, which can react with a nucleophilic side chains on the wild-type
channel. Light sensitivity was conferred to both cultured neurons and cerebellar
slices bathed in the different PALs. Because PALs target endogenous proteins, the
approach is applicable to systems where introduction of exogenous genes is diffi-
cult; however, it also leads to widespread photosensitivity based on the ion channel
targeting moiety. This limitation should be easily overcome using local application
of the PAL, local illumination, or utilization of a highly specific targeting moiety.
For example, injection of acrylamide PAL into the vitreous cavity of the mouse eye
(Polosukhina et al. 2012 ) restored the pupillary reflex in mutant mice similar to the
aforementioned LiGluR study (Caporale et al. 2011 ). In addition, injection of PAL
restored locomotory light behavior in mice lacking photoreceptors, suggesting a
new avenue for treatment of blinding diseases.
Bioreactive Tethers