Biomedical Engineering Reference
In-Depth Information
Probes for Inhibiting Neurons
NpHR was the fi rst molecule shown to inhibit neural activity (Zhang et al.
2007a
,
b
;
Han and Boyden
2007
) and is naturally expressed in the halobacterium
Natronomonas
pharaonis
(Schobert and Lanyi
1982
; Bamberg et al.
1993
). NpHR is a light-driven
pump, which actively pumps Cl ions into cells in response to yellow light at the
peak absorption wavelength of 570 nm. Like ChR2, NpHR utilizes retinal as its
chromophore and therefore can also be used in vertebrate systems without extra
cofactors. Substantial mutagenesis was required to achieve high levels of expression
in neurons. Enhanced halorhodopsin (eNpHR), a second-generation NpHR, pos-
sesses an endoplasmic reticulum export signal and thus displays improved translo-
cation to the plasma membrane (Zhao et al.
2008
; Gradinaru et al.
2008
).
Third-generation constructs (eNpHR3.0) have been generated to improve photocur-
rent increasing in membrane hyperpolarization over eNpHR, with additional
membrane- traffi cking sequences (Gradinaru et al.
2010
). Other proteins related to
bacteriorhodopsins have been discovered recently and can be used to inhibit neural
activity in response to light. Unlike NpHR, these proteins function as light-driven
proton pumps. Archaerhodopsin-3 (Arch) proteins are derived from
Halorubrum
sodomense
and allow near-100 % silencing of neurons in vivo in response to yellow
light, with an effi ciency comparable to that of eNpHR3.0 (Chow et al.
2010
). Two
other bacterial rhodopsins, Mac proteins from
Leptosphaeria maculans
and bacteri-
orhodopsin (BR) from
Halobacterium salinarum
(and its enhanced, second-
generation derivative, eBR), allow silencing of neurons in response to blue-green
light (Chow et al.
2010
; Gradinaru et al.
2010
). Therefore, hyperpolarizing optoge-
netic tools now exist that respond to blue- green and yellow light, allowing for
combinatorial dissection of two neural subtypes in the same preparation. High-
throughput genomic screens should reveal additional channels and thereby increase
the diversity of inhibitory optogenetic tools for future use.
Interestingly, in a recent set of experiments, a proton-pumping archeorhodopsin
was shown to allow high-speed imaging of individual action potentials. When a
single amino acid residue was mutated, the resulting structure prevented proton fl ow
across the membrane, allowing the channel to be used solely as an indicator. This
genetically encoded voltage indicator exhibited an approximately tenfold improve-
ment in sensitivity and speed over existing protein-based voltage indicators, with a
roughly linear twofold increase in brightness between −150 and +150 mV and a
sub-millisecond response time (Kralj et al.
2011
). Therefore, optogenetics allows
not only selective control of neural circuitry but also readout of its signals.
Probes for Manipulation of Intracellular Signaling
Neurons are also modulated by intracellular signaling events, initiated by cell sur-
face receptors that culminate in a change in neuronal electrical activity instead of
electrical signals through ion channels, as well as changes in secondary messenger
pathways leading to gene expression and downstream protein cascades. Because
