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The Development andApplication of OptogeneticsLief Fenno,1,2
Ofer Yizhar,1 and Karl Deisseroth1,3,41Department of
Bioengineering, 2Neuroscience Program, 3Departments of Psychiatry
andBehavioral Sciences, 4Howard Hughes Medical Institute, Stanford
University, Stanford,California 94305; email:
[email protected]
Annu. Rev. Neurosci. 2011. 34:389412
The Annual Review of Neuroscience is online
atneuro.annualreviews.org
This articles doi:10.1146/annurev-neuro-061010-113817
Copyright c 2011 by Annual Reviews.All rights reserved
0147-006X/11/0721-0389$20.00
Keywordschannelrhodopsin, halorhodopsin,
bacteriorhodopsin,electrophysiology
AbstractGenetically encoded, single-component optogenetic tools
have madea significant impact on neuroscience, enabling specific
modulation ofselected cells within complex neural tissues. As the
optogenetic toolboxcontents grow and diversify, the opportunities
for neuroscience con-tinue to grow. In this review, we outline the
development of currentlyavailable single-component optogenetic
tools and summarize the appli-cation of various optogenetic tools
in diverse model organisms.
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ContentsINTRODUCTION . . . . . . . . . . . . . . . . . .
390EARLY EFFORTS TOWARD
OPTICAL CONTROL . . . . . . . . . . . 390MICROBIAL OPSINS . . .
. . . . . . . . . . . . 391OPTOGENETIC TOOLS FOR
NEURONAL EXCITATION . . . . . 393OPTOGENETIC TOOLS FOR
NEURONAL INHIBITION . . . . . . 396OPTOGENETIC TOOLS FOR
BIOCHEMICAL CONTROL . . . . . 397DELIVERING OPTOGENETIC
TOOLS INTO NEURONALSYSTEMS . . . . . . . . . . . . . . . . . . .
. . . . . 398
TRANSGENIC ANIMALS. . . . . . . . . . . 399DEVELOPMENTAL AND
LAYER-SPECIFICTARGETING . . . . . . . . . . . . . . . . . . . .
399
CIRCUIT TARGETING . . . . . . . . . . . . 401LIGHT DELIVERY
AND
READOUT HARDWARE FOROPTOGENETICS. . . . . . . . . . . . . . . .
401
OPTOGENETICS IN DIVERSEANIMAL MODELS. . . . . . . . . . . . . .
. 402Caenorhabditis elegans . . . . . . . . . . . . . . . 402Fly .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
402Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . .
403Mouse. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
404Rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . 405Primate . . . . . . . . . . . . . . . . . . . . . . . . . .
. 405
OUTLOOK. . . . . . . . . . . . . . . . . . . . . . . . . .
405
INTRODUCTIONIn describing unrealized prerequisites for
as-sembling a general theory of the mind, FrancisCrick observed
that the ability to manipulateindividual components of the brain
wouldbe needed, requiring a method by which allneurons of just one
type could be inactivated,leaving the others more or less
unaltered(Crick 1979, p. 222). Extracellular electricalmanipulation
does not readily achieve true in-activation, and even electrical
excitation, whileallowing for temporal precision in
stimulatingwithin a given volume, lacks specificity for cell
type. However, pharmacological and geneticmanipulations can be
specific to cells withcertain expression profiles (in the best
case)but lack temporal precision on the timescale ofneural coding
and signaling.
Because no prior technique has achievedboth high-temporal and
cellular precisionwithin intact mammalian neural tissue, therehas
been strong pressure to develop a new classof technology. As a
result of these efforts, neu-rons now may be controlled with
optogeneticsfor fast, specific excitation or inhibition
withinsystems as complex as freely moving mammals[for example, with
microbial opsin methods,light-induced inward cation currents may
beused to depolarize the neuronal membrane andpositively modulate
firing of action potentials,while optical pumping of chloride ions
caninduce outward currents and membranehyperpolarization, thereby
inhibiting spiking(Figure 1)]. These optogenetic tools of
micro-bial origin (Figure 1) may be readily targetedto
subpopulations of neurons within hetero-geneous tissue and function
on a temporalscale commensurate with physiological ratesof spiking
or critical moments in behavioraltests, with fast deactivation upon
cessation oflight. With these properties,
microbe-derivedoptogenetic tools fulfill the criterion set forthby
Crick in 1979 (Deisseroth 2010, 2011).
EARLY EFFORTS TOWARDOPTICAL CONTROLThe microbial opsin approach
is heir to along tradition of using light as an interventionin
biology. With chromophore-assisted laserinactivation, light can be
used to inhibittargeted proteins by destroying them [whata
geneticist would call loss of function(Schmucker et al. 1994)];
conversely, lasers canbe used to stimulate neurons directly in a
waythat could be adapted (in principle) to controlfluorescently
labeled, genetically targeted cells[what a geneticist would call
gain of function(Fork 1971, Hirase et al. 2002)]. Next,
variouscascades of genes, and combinations of geneswith chemicals,
were tested as multicomponent
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Na+
Na+ Na+
Na+
Na+
Na+
K+
H+
H+
H+H+
H+
H+
K+K+
K+
K+
K+
Na+Na+
Na+ Na+ Ca2+
Ca2+Ca2+
Na+
Na+
ChR
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
ClCl
Cl
Cl
Cl
Cl
Cl
HR
[IP3][DAG]
[cAMP] [cAMP]
GsGq Gi
OptoXR
Figure 1Optogenetic tool families. Channelrhodopsins conduct
cations and depolarize neurons upon illumination (left).
Halorhodopsinsconduct chloride ions into the cytoplasm upon yellow
light illumination (center). OptoXRs are rhodopsin-GPCR (G
proteincoupledreceptor) chimeras that respond to green (500 nm)
light with activation of the biological functions dictated by the
intracellular loopsused in the hybrid (right).
strategies for optical control; rhodopsin andarrestin genes from
Drosophila photoreceptorswere combined to light-sensitize
neurons(Zemelman et al. 2002); ligand-gated channels,combined with
ultraviolet (UV)-light pho-tolysis of caged agonists, were
developed forDrosophila experiments (Lima & Miesenbock2005,
Zemelman et al. 2003); and UV lightisomerizable chemicals linked to
geneticallyencoded channels were employed in culturedcells and in
zebrafish (Banghart et al. 2004,Szobota et al. 2007, Volgraf et al.
2006). Theseefforts have been reviewed (Gorostiza &
Isacoff2008, Miesenbock & Kevrekidis 2005) andwhile elegant,
have thus far been found to belimited to various extents in speed,
targeting,tissue penetration, and/or applicability becauseof their
multicomponent nature. Here, wereview development and application
effortsfocused on the distinct single-component opto-genetic tools,
such as microbial opsins, over thepast six years since they were
first implemented.
MICROBIAL OPSINSSpecies from multiple branches of the an-imal
kingdom have evolved mechanismsto sense electromagnetic radiation
in their
environments. Likewise many microbes, in theabsence of complex
eye structures employedby metazoans, have developed
light-activatedproteins for a variety of purposes. For some,this
serves as a mechanism of homeostasis to re-main at a certain depth
in the ocean (Beja et al.2000, 2001); for others, this helps
maintainosmotic balance in a highly saline environment(Stoeckenius
1985). These and other diverseroles are, in many cases, fulfilled
by a familyof seven-transmembrane, light-responsiveproteins encoded
by opsin genes.
Opsin genes are divided into two distinctsuperfamilies:
microbial opsins (type I) and an-imal opsins (type II). Opsin
proteins from bothfamilies require retinal, a vitamin
Arelatedorganic cofactor that serves as the antenna forphotons;
when retinal is bound, the functionalopsin proteins are termed
rhodopsins. Retinalcovalently attaches to a conserved lysineresidue
of helix 7 by forming a protonatedretinal Schiff base (RSBH+). The
ionic envi-ronment of the RSB, defined by the residuesof the
binding pocket, dictates the spectraland kinetic characteristics of
each individualprotein. Upon absorption of a photon,
retinalisomerizes and triggers a sequence of confor-mational
changes within the opsin partner.
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The photoisomerized retinal is the triggerfor subsequent
structural rearrangements andactivities performed by these
proteins.
Although both opsin families encodeseven-transmembrane
structures, sequence ho-mology between the two families is
extremelylow; homology within families, however, ishigh (25%80%
residue similarity) (Man et al.2003). Whereas type I opsin genes
are found inprokaryotes, algae, and fungi (Spudich 2006),type II
opsin genes are present only in highereukaryotes and are
responsible mainly forvision (but also play roles in circadian
rhythmand pigment regulation) (Sakmar 2002,Shichida & Yamashita
2003). Type II opsingenes encode G proteincoupled receptors(GPCRs)
and, in the dark, bind retinal in the11-cis configuration. Upon
illumination, retinalisomerizes to the all-trans configuration
andinitiates the reactions that underlie the
visualphototransduction second messenger cascade.After
photoisomerization, the retinal-proteinlinkage is hydrolyzed; free
all-trans retinal thendiffuses out of the protein and is replaced
by afresh 11-cis retinal molecule for another roundof signaling
(Hofmann et al. 2009).
In contrast, type I opsins more typicallyencode proteins that
utilize retinal in theall-trans configuration, which
photoisomerizesupon photon absorption to the 13-cis con-figuration.
Unlike the situation with type IIrhodopsins, the activated retinal
molecule intype I rhodopsins does not dissociate from itsopsin
protein but thermally reverts to the all-trans state while
maintaining a covalent bondto its protein partner (Haupts et al.
1997).Type I opsins encode several distinct subfami-lies of
protein, discussed in more detail below.The central operating
principle of these ele-gant molecular machines [established for
thisbroad family of opsins since bacteriorhodopsin(BR) in 1971
(Oesterhelt & Stoeckenius 1971)and now including halorhodopsins
and chan-nelrhodopsins (Figure 1)] is their unitary na-ture. They
combine the two tasks of lightsensation and ion flux into a single
protein(with bound small organic cofactor), encodedby a single
gene. In 2005, one of these micro-
bial opsins was brought to neuroscience as thefirst
single-component optogenetic tool (Boy-den et al. 2005), and the
other microbial opsinsubfamilies followed close behind. For
example,channelrhodopsin-1 (ChR1) (Nagel et al. 2002)and
channelrhodopsin-2 (ChR2) (Nagel et al.2003) from Chlamydomonas
reinhardtii are blue-light-activated nonspecific cation channels.
Incommon with other type I opsins, these proteinsrequire retinal as
the photon-sensing cofactorto function. In response to light
stimulation, thechannel shuttles from the dark-adapted statethrough
a stereotyped progression of functionaland conformational states,
eventually (in the ab-sence of further light stimulation) reaching
thedark-adapted state once again. These differentstates, which all
have unique spectroscopic sig-natures, are collectively referred to
as the pho-tocycle, which (as for BR and halorhodopsinin earlier
work) has been extensively inves-tigated (Bamann et al. 2010,
Berndt et al.2010, Ernst et al. 2008, Hegemann et al. 2005,Ritter
et al. 2008, Stehfest et al. 2010, Stehfest& Hegemann
2010).
The size, kinetic properties, and wavelengthsensitivity of
photocurrents resulting from acti-vation of an individual protein
are a direct resultof its photocycle topology, ion selectivity,
andactivation/deactivation/inactivation time con-stants (Ernst et
al. 2008, Hegemann et al. 2005,Ritter et al. 2008). Typically, a
transient peakphotocurrent, evoked at the onset of light
stim-ulation, decays modestly to a steady-state pho-tocurrent even
in the presence of continuouslight, owing in part to the
desensitization ofa certain population of channels (Nagel et
al.2003). The desensitized population can recoverin the dark with a
characteristic time constanton the order of 5 seconds, giving rise
to a simi-lar peak photocurrent if a second light pulse isapplied
after sufficient time has elapsed (Nagelet al. 2003). The fraction
of desensitized pro-teins is crucial for determining the
reliability oflight stimulation during prolonged experiments(e.g.,
behavioral or long-term physiological ex-periments) where light is
applied for extendedperiods. This issue is addressed in more
detailbelow.
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OPTOGENETIC TOOLS FORNEURONAL EXCITATIONMany years passed after
the discovery of BRs,channelrhodopsins, and halorhodopsins, priorto
the development of optogenetics. As notedabove, optogenetics with
microbial opsins be-gan with a channelrhodopsin, introduced
intohippocampal neurons in 2005 (Boyden et al.2005) where it was
found to confer millisecond-precision control of neuronal spiking.
A num-ber of additional reports followed over the nextyear (Bi et
al. 2006, Ishizuka et al. 2006, Liet al. 2005, Nagel et al. 2005).
Moreover, thisturned out to be a single-component system;through a
remarkable twist of nature, investi-gators found that sufficient
retinal is presentin mammalian brains (and as later established,in
all vertebrate tissues tested thus far) to en-able functional
expression of these optogenetictools as single components, in the
absence ofany added chemical or other gene (Deisserothet al. 2006,
Zhang et al. 2006). The optoge-netic toolbox has been vastly
expanded sincethe original 2005 discovery to include dozensof
single-component proteins activated by var-ious wavelengths of
light, with various ion con-ductance regulation properties that
operate inneurons over a wide range of speeds (from mil-liseconds
to tens of minutes), enabling broadexperimental configurations and
opportunities.
Initial improvements in ChR2 were carriedout in an incremental
fashion and focusedon improving expression and photocurrentin
mammalian systems. Human codon opti-mization for improved
expression of ChR2(Boyden et al. 2005, Gradinaru et al. 2007)
wascombined with substitution of histidine forarginine at position
134 to increase steady-statecurrent size, although the mechanism of
thelatter effect (delayed channel closure) alsosignificantly
impaired temporal precision andhigh-speed spiking (ChR2-H134R;
Nagel et al.2005, Gradinaru et al. 2007). Better solutionswere
needed, and further diversification of theoptogenetic toolbox via
mutational engineer-ing has proven to be challenging but
highlyproductive (Figure 2).
First, because many microbial tools do notexpress well in
mammalian neurons, generalstrategies for enabling mammalian
expressionwere required. Membrane-trafficking modi-fications turned
out to be crucial, beginningwith the observation in 2008 that
endo-plasmic reticulum (ER)-export motifs werehelpful for achieving
high, safe expression ofhalorhodopsins (Gradinaru et al. 2008,
Zhaoet al. 2008), a principle that turned out to beextendable [to
other classes of membrane-trafficking motif (Gradinaru et al.
2010)] andgeneralizable [to most microbial opsins tested(Gradinaru
et al. 2010)]. Next, chimera strate-gies (using hybrids of ChR1 and
ChR2) werefound to be helpful in giving rise to improvedexpression
and spiking properties for channel-rhodopsins (Lin et al. 2009,
Wang et al. 2009).
Finally, much subsequent opsin engineer-ing for optogenetics was
carried out on the ba-sis of hypothesized ChR structures. To
date,there has been no reported crystal structureof any excitatory
optogenetic tools, althoughstructures exist for halorhodopsin and
for theproton pump BR (Luecke et al. 1999a,b), whichcan function to
inhibit neurons when heterol-ogously expressed (Gradinaru et al.
2010); pro-ton conductance is a property shared by ChR2,whether
through channel or possible pumpingmechanisms (Feldbauer et al.
2009). Capital-izing upon homology between BR and ChRs,we have
introduced a number of mutations thatmodify various properties of
the opsins (dis-cussed below) and have described a frameworkby
which these improvements can be applied toother novel opsins
[indeed, this direction hascome full circle, with insights from
ChRs nowused to improve the optogenetic function of BRitself
(Gradinaru et al. 2010)].
These structural hypothesis-based opsinengineering efforts were
spurred by inherentlimitations of the channelrhodopsin
system(Figure 2). Specifically, the deactivation timeconstant of
ChR2 upon cessation of light(1012 ms in neurons) imposed a limit
ontemporal precision, leading in some cases toartifactual
multiplets of spikes after even singlebrief light pulses (as well
as a plateau potential
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Blue (off /peak activation wavelength)
ChR2
ChIEF
0 10 155 20 25off (ms)
~10 ms/470 nm
ChR2 (H134R) 18 ms/470 nm
~10 ms/450 nm
Blue ChETA (off /peak activation wavelength)
T159C/E123T
0 2 4 6 8off (ms)
E123A 4 ms/470 nm
E123T 4.4 ms/500 nm8 ms/500 nm
Red-shifted (off /peak activation wavelength)
C1V1
0 50 100 150off (ms)
120 ms/540 nm
VChR1 133 ms/545 nm
Red-shifted ChETA (off /peak activation wavelength)
0 10 20 30 40 50off (ms)
38 ms/545 nmC1V1 (E162T/E122T)C1V1 (E162T) 50 ms/535 nm
Bistable (off /peak activation wavelength/peak inactivation
wavelength)
0 3 6 9 12 15 18 21 24 27 30off (min)
C128S 1.7 min/470 nm/560 nmChR2 0.000167 min
D156A 6.9 min/470 nm/590 nm
C128S/D156A 29 min/445 nm/590 nm
Biochemical signaling (off /peak activation wavelength)
1AR
0 1 2 203 off (s)
3 s/500 nm
2AR 0.5 s/500 nm
b-PAC 20 s/441 nmRh-CT 3 s/485 nm
Inhibitory (off /peak activation wavelength)
ArcheBR
0 2015105off (ms)
18.9 m/560 nm
eNpHR3.0 4.2 ms/590 nm
9 ms/566 nm
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when pulsing blue light at frequencies above 40hz; the next
pulse of light would occur before allthe ChRs had deactivated,
leading eventuallyto failed deinactivation of native
voltage-gatedsodium channels and thus missed spikes andfurther loss
of fidelity). The latter problem wascompounded by the
desensitization of ChRitself even in the presence of light, leading
tofurther missed spikes late in trains. Addressingpart of this
challenge, the chimeric opsins notedabove (e.g., ChIEF; Lin et al.
2009), demon-strated reduced desensitization in culturedneurons,
allowing more robust spiking overtrains in culture as well as
stronger currents. Inanother approach addressing both
desensitiza-tion and deactivation, considering the crystalstructure
of BR led to modification of the coun-terion residue E123 of ChR2
to threonine oralanine; the resulting faster opsin is referred toas
ChETA (Figure 2) (Gunaydin et al. 2010).This substitution
introduced two advantagesover wild-type ChR2. First, it reduced
desen-sitization during light exposure, with the resultthat light
pulses late in high-frequency trainsbecame as likely as early light
pulses to drivespikes (a very important property referred to
astemporal stationarity). Second, it destabilizedthe active
conformation of retinal, speedingspontaneous isomerization to the
inactive stateafter light-off and thus closing the channelmuch more
quickly after cessation of light thanwild-type or improved ChR2
variants. Theresulting functional consequences of ChETAmutations
are temporal stationarity, reducedextra spikes, reduced plateau
potentials, andimproved high-frequency spike followingat 200 Hz or
more over sustained trains,even within intact mammalian brain
tissue(Gunaydin et al. 2010).
Whereas many experimental designs em-ploy optogenetic tools to
initiate precise spik-ing, alternative paradigms may instead
requirethe researcher to simply alter the excitabilityof a target
neuronal population. Indeed, it isoften important to bias the
activity of a par-ticular neuronal population without
specificallydriving action potentials or synchronous ac-tivity. To
facilitate experiments examining al-tered excitability, Berndt et
al. (2009) devel-oped the step-function opsins (SFOs). SFOs area
family of ChR mutants that display
bistablebehaviororders-of-magnitude prolonged ac-tivity after
termination of the light stimulusfirst instantiated as mutations in
position 128in ChR2 (cysteine to serine, alanine, or threo-nine).
Again based on homology between ChR2and BR (Peralvarez-Marin et al.
2004), we mu-tated this residue to manipulate the interac-tion
between the opsin backbone and the cova-lently bound retinal photon
sensor. In contrastto ChETA, the SFO mutations are designedto
stabilize the active retinal isomer, the func-tional consequence of
which is prolonging theactive state of the channel even after
light-off.SFOs have inactivation time constants on theorder of tens
of seconds or more instead of mil-liseconds (Figure 2) and can be
activated by asingle 10-ms pulse of blue light (Berndt et al.2009).
The SFOs can also be deactivated by apulse of yellow light; the
yellow pulse drivesisomerization of retinal back to the
noncon-ducting state. A subsequently engineered SFO,the ChR2(D156A)
opsin (Bamann et al. 2010),displays an even longer inactivation
time con-stant, which in our hands approaches eight min-utes. One
potential noted use of opsins withextended time constants could be
for scanningtwo-photon stimulation paradigms (Rickgauer
Figure 2Spectral and kinetic diversification of optogenetic
tools. Deactivation time constants (off ) and approximate peak
activation/inactivationwavelengths are shown for blue
lightactivated opsins, blue ChETAs (including E123T/T159C) (Berndt
et al. 2011, Mattis et al. 2011),red-shifted opsins, red-shifted
ChETAs, bistable (SFO) opsins, and modulatory/inhibitory tools in a
compact look-up table. ChR2 islisted among the bistable group for
scale purposes only. Where precise values are not available, decay
kinetics were measured (courtesyof J. Mattis, personal
communication) or estimated from published traces; all values were
recorded at room temperature (except foroptoXRs measured at 37C),
with substantial acceleration in kinetics (50%) expected for all at
37C.
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& Tank 2009), during which it would be help-ful to have
persistent accumulating activity as asmall two-photon spot scans a
cell or tissue ofinterest.
We have now engineered a third classof SFO by combining the D156
and C128mutations to produce a ChR2 variant that hasa spontaneous
deactivation time constant ap-proaching 30 min; this stabilized SFO
(SSFO)(O. Yizhar, L. Fenno, M. Prigge, K. Stehfest,J. Paz, F.
Schneider, S. Tsunoda, R. Fudim, C.Ramakrishnan, J. Huguenard, P.
Hegemann& K. Deisseroth, submitted) induces peakcurrents of
>200 pA. An advantage of havingan opsin with such a long time
constant is theability to conduct behavioral protocols in
theabsence of tethered external light delivery de-vices (e.g.,
optical fibers). Because a single pulseof blue light is sufficient
to induce activity for atime period extending beyond that of most
be-havioral paradigms, the fiber may be removedbefore commencing
the experiment. Just aswith the original SFO proteins, SSFO may
beinactivated by yellow light, allowing for precisecontrol of
network dynamics. A final advantageof the SFOs (which scales with
their kinetic sta-bility) is orders-of-magnitude greater light
sen-sitivity of transduced cells, particularly for longlight
pulses, a direct result of the photon inte-gration bestowed by
their prolonged deactiva-tion time constant. This property renders
SFOsespecially attractive as minimally invasive toolsfor
stimulating large brain regions (for example,in primate studies)
and deep within tissue.
Separate from, but synergistic with, molec-ular engineering is
the systematic genomicidentification of novel opsins. Adapting
novelopsins activated by red-shifted wavelengthscould enable
control of two separate popu-lations of circuit elements within the
samephysical volume. To this end, we launched ge-nomic search
strategies that led to identificationof an opsin from Volvox
carteri (VChR1) (Zhanget al. 2008), which shares homology with
ChR2and similarly functions as a cation channel.In contrast to
ChR2, VChR1 is activated byred-shifted light. However, the
relatively smallcurrents due to low expression in mammalian
neurons, as described in the initial report,have hampered in
vivo adaptation of VChR1,even after codon optimization. To this
end, wehave engineered a chimeric opsin, C1V1 (O.Yizhar, L. Fenno,
M. Prigge, K. Stehfest, J.Paz, F. Schneider, S. Tsunoda, R. Fudim,
C.Ramakrishnan, J. Huguenard, P. Hegemann& K. Deisseroth,
submitted), composed ofthe first two and one-half helices of ChR1(a
poorly expressing relative of ChR2 fromthe same organism) (Nagel et
al. 2002) andthe last four and one-half helices of VChR1.The
resulting tool retains the red-shiftedactivation spectrum of VChR1,
but withnanoampere-scale currents that exceed thoseof ChR2. The
large current of C1V1 allowsuse in vivo and also use of off-peak
(redder)light wavelengths, together enabling trulyseparable control
of multiple populations ofneurons when used in conjunction with
ChR2(O. Yizhar, L. Fenno, M. Prigge, K. Stehfest,J. Paz, F.
Schneider, S. Tsunoda, R. Fudim, C.Ramakrishnan, J. Huguenard, P.
Hegemann& K. Deisseroth, submitted). In addition
tocombinatorial experiments with ChR2, toolswith red-shifted
activation wavelengths suchas C1V1 are also more amenable to use
withsimultaneous imaging of genetically encodedcalcium indicators,
such as GCaMP variants(Hires et al. 2008, Zhang & Oertner
2007).
OPTOGENETIC TOOLS FORNEURONAL INHIBITIONComplementing these
tools for precise controlof neural excitation, certain
light-activated ionpumps may be used for inhibition, althoughthus
far only one ion pump has shown efficacyat modulating behavior in
mammals (Tye et al.2011, Witten et al. 2010, Zhang et al.
2007a):the ER trafficking-enhanced version of ahalorhodopsin called
NpHR (Gradinaru et al.2008, 2010; Zhang et al. 2007a) derived
fromthe halobacterium Natronomonas pharaonis. Inthe context of
optogenetic application, this yel-low lightactivated electrogenic
chloride pumpacts to hyperpolarize the targeted neuron
uponactivation (Figure 1) (Zhang et al. 2007a).
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Unlike the excitatory channelrhodopsins,NpHR is a true pump and
requires constantlight to move through its photocycle.
Althoughoptogenetic inhibition with NpHR was shownto operate well
in freely moving worms (Zhanget al. 2007a), mammalian brain slices
(Zhanget al. 2007a), and cultured neurons (Han &Boyden 2007,
Zhang et al. 2007a), several yearspassed before final validation of
this (or any) in-hibitory optogenetic tool was obtained by
suc-cessful application to intact mammals (Tye et al.2011, Witten
et al. 2010) because of membranetrafficking problems that required
additionalengineering (Gradinaru et al. 2008, 2010; Zhaoet al.
2008). Indeed, a number of modificationsto NpHR were required to
improve its func-tion, initially codon-optimizing the
sequencefollowed by enhancement of its subcellu-lar trafficking
(eNpHR2.0 and eNpHR3.0)(Gradinaru et al. 2008, 2010), which
resultedin improved membrane targeting and highercurrents suitable
even for use in human tissue(Busskamp et al. 2010), as well as
activation withred-shifted wavelengths at the infrared border(680
nm). Kouyama et al. (2010) publishedthe 2.0A crystal structure of
halorhodopsinand illustrate that this protein has a highdegree of
homology within the retinal bindingpocket with the proton pump BR.
The two areproposed to distribute charge and store energyfrom
absorbed photons in similar ways.
The diversification of the inhibitory opsintoolbox has been
guided by bioinformatics ap-proaches to screen nature for novel
inhibitoryion pumps with desirable properties, just as hadbeen
successful previously for excitatory opsins(Zhang et al. 2008). In
2010, we and othersexplored the use of proton pumps (eBR, Mac,and
Arch) as optogenetic tools (Gradinaruet al. 2010, Chow et al.
2010), finding evidencefor robust efficacy but leaving open
questionsof long-term tolerability and functionality
ofproton-motive pumps in mammalian neurons.A major concern is the
extent to which pump-ing of protons into the extracellular
space(especially in juxtamembranous compartmentsdifficult to
visualize or measure) could havedeleterious or
noncell-type-specific effects on
local tissue, which could show up as light-induced inhibition
affecting all recorded units(more units than expected for a given
transduc-tion efficiency) or with a slightly slower timecourse than
expected for the fast proton pumps.
Although the proton pumps must be treatedwith caution until
these issues are addressed,many opportunities exist; indeed, we
haveimproved the ability of proton pumps to hy-perpolarize neurons
following a methodologysimilar to that used for improving
NpHR(Gradinaru et al. 2010), although none of theproton pumps yet
described is as kinetically sta-ble or as potent as eNpHR3.0,
especially at thesafe light levels (
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Rhodopsin, the light-sensing protein in themammalian eye, is
both an opsin, in that it iscovalently bound to retinal and its
function ismodulated by the absorption of photons, and aGPCR, in
that it is coupled on the intracellularside of the membrane to a
G-protein, trans-ducin. Virtually all neurons can communicatevia
GPCRs, which of course respond notonly to neuromodulators from
dopaminergic,serotonergic, and adrenergic pathways, but alsoto fast
neurotransmitters such as glutamateand GABA. Building both on our
finding thatadequate retinal is present within mammalianbrain
tissue (Deisseroth et al. 2006, Zhanget al. 2006) and on a long
history of elegantGPCR structure-function work from
Khorana,Kobilka, Caron, Lefkowitz, and others, wehave determined
that GPCRs can be convertedinto light-activated regulators of
well-definedbiochemical signaling pathways that functionwithin
freely moving mammals. These pro-teins are referred to as optoXRs
(Airan et al.2009). OptoXRs allow for
receptor-mediatedintracellular signaling with temporal
resolutionsuitable for modulation of behavior in freelymoving mice
(Airan et al. 2009). OptoXRs aremodulated by 500-nm light and now
include thealpha-1 and beta-2 adrenergic receptors (Airanet al.
2009), which are coupled to Gq and Gs sig-naling pathways,
respectively, and the 5-HT1areceptor, which is Gi/o coupled (Oh et
al. 2010).
Optical control over small GTPases wasnext achieved in cultured
cells by several differ-ent laboratories (Levskaya et al. 2009, Wu
et al.2009, Yazawa et al. 2009) using optically mod-ulated
protein-protein interactions; althoughthese have not yet been shown
to express or dis-play single-component functionality in
freelymoving mammals, such capability is plausiblewhere flavin or
biliverdin chromophores arerequired and present. Finally,
investigatorshave recently described microbial adenylylcyclases
with lower dark activity than earliermicrobial cyclases, and
because they employ aflavin chromophore, these tools appear
suitablefor single-component optogenetic control(Ryu et al. 2010,
Stierl et al. 2010). Together,these experiments have extended
optogenetic
capability to essentially every cell type, even innonexcitable
tissues, in biology.
DELIVERING OPTOGENETICTOOLS INTO NEURONALSYSTEMSViral expression
systems have the dual advan-tages of fast/versatile implementation
and highinfectivity/copy number for robust expressionlevels.
Cellular specificity can be obtained withviruses by specific
promoters (if small, specific,and strong enough), by spatial
targeting of virusinjection, and by restriction of opsin
activationto particular cells (or projections of specificcells) via
targeted light delivery (Zhang et al.2010, Diester et al. 2011).
Lenti and adeno-associated (AAV) viral vectors have been
usedsuccessfully to introduce opsins into the mouse,rat, and
primate brain (Zhang et al. 2010). Ad-ditionally, these have been
well tolerated andhighly expressed over long periods of time withno
reported adverse effects. Lentivirus is easilyproduced using
standard tissue culture tech-niques and an ultracentrifuge (see
Zhang et al.2010 for protocol). AAV may be produced ei-ther by
individual laboratories or through coreviral facilities. Neither
AAV nor lentivirus werefound to be highly expressed in the
zebrafish,for which Sindbis and rabies are more effective(Zhu et
al. 2009). Viruses have been used totarget (among other cells)
hypocretin neurons(Adamantidis et al. 2007), excitatory
pyramidalneurons (Lee et al. 2010, Sohal et al. 2009,Zhang et al.
2007a), and astroglia (Gourine et al.2010, Gradinaru et al. 2009).
For example, onegroup (Gourine et al. 2010) recently describedthe
use of AAV-delivered ChR2 to controlastroglial activity in the
brain stem of mice andto dissect a mechanism by which astroglia
cantransfer systemic information from the bloodto neurons
underlying homeostasis, in this casedirectly modulating neurons
that manipulatethe rate of respiration. However, a major down-side
of viral expression systems is a maximumgenetic payload length;
only promoter frag-ments that are small (less than 4 kb),
specific,and strong may be used, and these are rare. This
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limitation may be skirted using Cre-driveranimals and
Cre-dependent viruses, discussedbelow.
TRANSGENIC ANIMALSThe use of transgenic or knock-in animals
ob-viates viral payload limitations and allows fortighter control
of transgene expression usinglarger promoter fragments or indeed
the en-dogenous genome in full via knock-in. Thefirst transgenic
opsin-expressing mouse line wasgenerated using the Thy1 promoter
(Arenkielet al. 2007, Zhao et al. 2008), with widespreadexpression
throughout neocortical layer 5 pro-jection neurons as well as in
some subcorticalstructures (Arenkiel et al. 2007). This mouseline
has been widely used, for example, to exam-ine the roles of
inhibitory neurons on corticalinformation processing (Sohal et al.
2009) andthe mechanism of action of deep brain stimu-lation for
Parkinsons disease (Gradinaru et al.2009). Several other groups
have subsequentlyalso generated transgenic mouse lines
directlyexpressing opsin genes (Hagglund et al. 2010,Katzel et al.
2010, Thyagarajan et al. 2010).
Caveats to using transgenic mouse linesto directly express
optogenetic tools includethe time, effort, and cost associated with
theirproduction, validation, and maintenance. Toenable widespread
use of the latest optoge-netic tools, investigators have designed
opsin-delivering viruses for which opsin expression isdependent on
the coexpression of Cre recom-binase (Figure 3). This doublefloxed
invertedopen-reading-frame (DIO; reviewed in Zhanget al. 2010)
strategy (Atasoy et al. 2008, So-hal et al. 2009, Tsai et al. 2009)
situates theopsin gene (inhibitory or excitatory) in the in-verted
(meaningless) orientation, but the geneis flanked by two sets of
incompatible Cre re-combinase recognition sequences (Sohal et
al.2009, Tsai et al. 2009). The recombinase recog-nition sequences
are placed such that in thepresence of Cre, the ORF is inverted
insteadof being excised. Reversing the sequence thenallows one of
the Cre recognition sites to be ex-cised (Figure 3), locking the
reading frame into
the correct direction and allowing for strongexpression of the
opsin with (for example) theelongation factor 1-alpha (EF1)
promoter.The specificity of this gene expression can thencome (for
example) from the targeted expres-sion of Cre in driver rodent
lines in whichCre is controlled with high specificity in thecontext
of very large chromosomal promoter-enhancer regions; the DIO
strategy thus en-ables versatile and widespread use of
optoge-netics with the many (and growing numberof ) experimental
systems selectively expressingCre recombinase (Geschwind 2004; Gong
et al.2003, 2007; Heintz 2004). This strategy hasbeen used recently
in many systems, for exam-ple to target dopamine-1 (D1) or
dopamine-2(D2) receptorexpressing neurons of the stria-tum via
transgenic D1-Cre and D2-Cre mouselines, to examine the effects of
their stimulationin the classic direct/indirect-movement path-ways
(Kravitz et al. 2010). With these sameCre lines, Lobo and
colleagues (2010) exam-ined the roles of nucleus accumbens D1 andD2
neurons in modulating cocaine reward. TheCre-dependent optogenetic
system allowed forthe first time a direct examination of the
re-lationship between neuronal activity of specificneuronal
populations and animal behavior, thuspaving the way for a deeper
understanding ofdiseases such as Parkinsons disease, depression,and
substance abuse.
DEVELOPMENTAL ANDLAYER-SPECIFIC TARGETINGThe ability to target
specific neocortical layershas been a long-sought goal of
neuroscience;this can now be achieved either with layer-specific
Cre driver lines or with developmentaltargeting strategies such as
in utero electropo-ration (IUE). As a result, multiple
laboratorieshave now successfully teased apart the role
oflayer-specific neurons in behavioral paradigmsand network
dynamics. Optogenetic toolshave been well tolerated when
electroporatedin utero into mouse embryos (Adesnik &Scanziani
2010, Gradinaru et al. 2007, Lewiset al. 2009, Petreanu et al.
2007); IUE may
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+ Cre Cre
+ Cre Cre
Perc
enta
ge
of m
axim
um (%
)Pe
rcen
tage
of
max
imum
(%)
YFP intensity+Cre Cre
b c
eYFP
Parvalbumin
Transgenicparvalbumin::Cre
Overlay
30 m
a
Cre+
Cre+
Cre+
Cre+
Cre+
Cre
Cre
Cre
Viralinjection
eYFP
loxP loxP
ChR2eYFPEF1
ChR2 eYFPEF1
ChR2eYFPEF1
ChR2 eYFPEF1
ChR2 eYFPEF1
ChR2 eYFPEF1
lox2722
lox2722loxP
lox2722
ReversibleCre-mediated
recombination
PermanentCre-mediated
lox site excision
d Floxed stop
loxP loxP
ITR
hGHpolyA
hGHpolyA
ITR3X polyA WPRE
Double-floxed inverse ORFeITR
100 um
100
80
60
40
20
01010 1020 1030
100
80
60
40
20
01010 1020 1030
YFP intensity
ITRWPRE
loxP loxP
lox2722 lox2722
Figure 3Low-leak Cre-dependent expression using the doublefloxed
inverted open-reading-frame (DIO) strategy. The combination of
atransgenic mouse expressing Cre recombinase in specific neuronal
subtypes and the injection of a virally encoded DIO opsin (a)
resultsin the physical inversion of the open reading frame (ORF) in
only that population (b,c), which may be transient and revert back
to theoriginal state or undergo further recombination to be
permanently anchored in the sense direction, resulting in
functional expression ofthe opsin (c). The DIO strategy may be
contrasted with the lox-stop-lox (floxed STOP) strategy (d,e). In
the absence of Crerecombinase, lox-stop-lox (d ) allows for some
level of expression leak as assayed by both enhanced yellow
fluorescent protein (eYFP)expression and fluorescence-activated
cell sorting (FACS) analysis. Because the ORF of an opsin in DIO
configuration encodesnonsense (e), there is no functional
expression in the absence of Cre recombinase. Adapted with
permission from Sohal et al. (2009) andF. Zhang and K.
Deisseroth.
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be used to target specific layers of cortex byincorporating the
DNA (with no promoter sizelimit) into neurons being born during a
specificembryonic stage. A major advantage of usingIUE or
transgenic mice over viral infection isthat opsins are expressed at
the time of birth,allowing electrophysiological researchers
toharvest acute slices at a younger stage. Counter-acting this
advantage is the fact that transgenicanimals typically express
lower levels of opsins,likely owing to the reduced gene copy
number.
CIRCUIT TARGETINGAnother generalizeable strategy for targeting
isreferred to as projection targeting, which cap-italizes on the
efficient membrane traffickingof engineered opsin gene products,
especiallydown axons to axon terminals. Light can bedelivered not
to somata but to axons, therebyrecruiting cells defined by virtue
of theirwiring without any genetic information aboutthe downstream
target required (Gradinaruet al. 2007, 2009; Lee et al. 2010;
Petreanu et al.2007). Neurons may also be targeted by projec-tion
using viruses that transduce axon terminals,such as herpes simplex
virus (HSV) familyviruses, certain serotypes of AAV, or
pseudo-typed lentiviruses. Trans-synaptic targetingmay be achieved
by exploiting the transcellulartrafficking of, for example, the
wheat germ ag-glutinin (WGA) peptide sequence (Gradinaruet al.
2010), which can deliver Cre recombinaseto the site of a second
Cre-dependent virusinjection. Combination strategies are also
pos-sible; by crossing transgenic Drd2-GFP micewith mice expressing
Cre under the controlof Emx1, then injecting DIO-ChR2-mCherryinto
cortex, Higley & Sabatini (2010) were ableto localize synapses
originating from cortex(red projections) onto neurons expressing
thedopamine-2 receptor (D2) in striatum (greencell bodies) and use
brief pulses of blue lightto elicit synaptic activity onto the D2
neurons.Combining optogenetic manipulation of thesynapse with
pharmacology and two-photonglutamate uncaging allowed the
investigatorsto elaborate precisely upon the role of D2 re-ceptors
in glutamatergic synaptic transmission.
A noteworthy method pioneered by theCallaway group (Wickersham
et al. 2007a,b)using a glycoprotein-deficient pseudotypedrabies
virus is yet another technique formonosynaptic circuit tracing.
Rabies virusis well known to travel trans-synapticallyfrom neuron
to neuron; the virus used inthis technique is not able to produce
viablepackaged copies of itself after moving trans-synaptically and
thus will be stopped after onesynapse jump. Applying this method
enablestracing of all neurons synaptically connectedto a single
neuron of interest. Two plasmids,one containing the
glycoprotein-deficientrabies payload and another containing
theglycoprotein, are coelectroporated into a singleneuron in vivo.
This neuron is then able toproduce competent rabies virus; the
payload,however, does not encode the coat protein. Inthis case, the
virus is stuck after moving onesynapse. By using red and green
fluorophoresin the two components, the targeted neuronand its
synaptic partners may be identified.This system has not yet been
integrated withoptogenetics, however, and the extremely highlevels
of expression resulting from rabies virusexpression may result in
toxicity incompatiblewith typical optogenetic experiments.
LIGHT DELIVERY ANDREADOUT HARDWARE FOROPTOGENETICSOptogenetics
fundamentally relies on light-delivery technology, the development
of whichhas led to improved precision of modulationboth in vitro
and in vivo. In vitro, optogenetictools are typically activated
with filtered lightfrom mercury arc lamps (e.g., Berndt et al.2009,
Boyden et al. 2005, Gunaydin et al.2010), lasers (Cardin et al.
2010, Cruikshanket al. 2010, Kravitz et al. 2010, Petreanu et
al.2009), light-emitting diodes (LEDs) (e.g.,Adesnik &
Scanziani 2010; Grubb & Burrone2010a,b; Wang et al. 2009), or
LED arraysfor multisite stimulation (Grossman et al.2010). In vivo,
stimulation of behaving animalshas been conducted mostly with laser
lightdelivered to the transduced tissue via optical
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fibers inserted through chronically implantedcannulas
(Adamantidis et al. 2007, Aravaniset al. 2007, Zhang et al. 2010)
or with fiber-coupled high-power LEDs (Wang et al. 2010).The
chronic delivery of light using implantedinfrared-triggered LEDs
(Iwai et al. 2011) is inthe early developmental stages but promises
toopen a new direction in optogenetic research.
To achieve rich readouts from optogeneti-cally controlled
tissue, major effort has been di-rected toward generating
electrophysiologicalsystems that combine high-density
single-unitrecordings with optogenetic stimulation inmice and other
organisms. The first readoutsfrom in vivo optogenetic modulation
wereobtained in anesthetized animals using a devicecomposed of a
fiberoptic cable integrated witha tungsten electrode (Gradinaru et
al. 2007),called an optrode (Cardin et al. 2009; Gradi-naru et al.
2007, 2009; Sohal et al. 2009; Zhanget al. 2009). More advanced
strategies haveemerged recently, employing silicone
multisiteelectrodes (Royer et al. 2010) and movabletetrode arrays
combined with optical fibers formore flexible interrogation of
neural activity invivo (Lima et al. 2009). Two-photon imagingis
another avenue with which optogenetics maybe integrated to
stimulate and record neuralactivity simultaneously. Several studies
havemade progress toward this type of experiment(Andrasfalvy et al.
2010, Mohanty et al. 2008,Papagiakoumou et al. 2010, Rickgauer
& Tank2009, Zhu et al. 2009); it seems that the majorlimitation
that hampers optogenetic activationwith two-photon approaches is
the combi-nation of rapidly decaying opsin-mediatedphotocurrents in
the setting of typical slow2P raster-scanning techniques.
Modifyingthe raster scan paradigm (Rickgauer & Tank2009) or
modulating the laser light such thatfast activation is possible
across wider regions(i.e., an entire cell soma; Papagiakoumouet al.
2010) can address this problem (Shoham2010). Appropriately rich
readouts, when com-bined with optogenetic inputs, will
powerfullyfacilitate fundamental studies regarding theorganization
and function of intact, complexneural networks.
OPTOGENETICS IN DIVERSEANIMAL MODELS
Caenorhabditis elegans
In transgenic nematodes harboring the chan-nelrhodopsin gene, it
is possible to controlmuscle wall motor neuron and mechanosen-sory
neuron activity (Nagel et al. 2005). Zhangand colleagues (2007a)
controlled body wallmuscle contraction bidirectionally with ChR2and
NpHR, demonstrating the power of com-binatorial optogenetics. This
concept has beenbuilt on with the description of
three-colorLCD-based multimodal light delivery (Stirmanet al. 2011)
and digital micromirror device(DMD)/laser light delivery (Leifer et
al. 2011),each coupled with tracking software for use withthe
behaving specimen. The facility of quanti-fying body-wall
contraction and elongation inC. elegans has enabled large-scale
investigationof various mutant strains for synaptic proteindefects
(Liewald et al. 2008, Stirman et al. 2010)and nicotinic
acetylcholine receptor function(Almedom et al. 2009). Finally, C.
elegans wasalso used for combined light-based stimulationand
readout of neural activity (Guo et al. 2009,Tian et al. 2009),
fulfilling the promise ofall-optical physiological experiments
usingoptogenetic tools and genetically encodedactivity sensors
(Scanziani & Hausser 2009).
FlyFly lines expressing upstream activation se-quence (UAS):ChR2
(Zhang et al. 2007b) havebeen used to investigate the neuronal
basis ofthe nociceptive response (Hwang et al. 2007)and
appetitive/aversive odorant learning at thereceptor (Bellmann et
al. 2010) or neurotrans-mitter (Schroll et al. 2006) level and to
rescuephotosensory mutants (Xiang et al. 2010). Ad-ditionally,
Hortstein et al. (2009) demonstratedGal4/UAS targeting of ChR2 to
the larval neu-romuscular junction system. Creative uses
ofoptogenetic tools in Drosophila include validat-ing neurons
identified in a screen to probethe proboscis extension reflex
(Gordon & Scott
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2009), driving monoamine release to validatefast-scanning cyclic
voltammetry detection ofserotonin and dopamine (Borue et al. 2009),
andinvestigating the innate escape response (Zim-mermann et al.
2009). Special considerationsare required for this model organism
(Pulveret al. 2009). Unlike mammals, flies and wormsdo not possess
levels of endogenous retinal suf-ficient for the function of
optogenetic tools,but food supplement can provide sufficientretinal
to drive ChR2 function (Xiang et al.2010).
Flies also possess innate behavioral re-sponses to blue light
that are developmentallydependent (Bellmann et al. 2010, Pulveret
al. 2009, Suh et al. 2007, Xiang et al.2010), complicating
behavioral studies usingopsins with blue activation spectra.
Thisconfound may be partially rectified using flylines without
vision, such as those lacking thenorpA gene (Bellmann et al. 2010),
althoughnorpA-deficient lines remain sensitive to bluelight (Xiang
et al. 2010). These issues could,in principle, be circumvented with
red-shiftedoptogenetic tools for excitation and
inhibition.Complementing eNpHR3.0 for red-shiftedinhibition, we
have developed an optogenetictoolset for potent red-shifted
excitation (O.Yizhar, L. Fenno, M. Prigge, K. Stehfest, J.Paz, F.
Schneider, S. Tsunoda, R. Fudim, C.Ramakrishnan, J. Huguenard, P.
Hegemann &K. Deisseroth, submitted). These have yet tobe tested
in Drosophila, but their green peak ac-tivation wavelength is
outside the range of keyDrosophila photosensory proteins (Xiang et
al.2010); moreover, spiking may be driven withup to 630 nm light,
improving the potential fordeep-penetrating excitation (Pulver et
al. 2009).
ZebrafishThe short generational time and easy inte-gration of
foreign DNA into zebrafish arecomplemented by ease of optogenetic
manip-ulation owing to transparency of the organism(McLean &
Fetcho 2011, White et al. 2008).The first use of optogenetic tools
in zebrafish(Douglass et al. 2008) appeared in a study
examining the role of somatosensory controlof escape behavior.
The use of ChR2 to drivesingle spikes in a genetically defined
populationduring the course of movement took advantageof a number
of properties of optogenetic toolsnot available with traditional
pharmacologicalor electrophysiological methods. Neuronswere
stimulated with a simple setup combin-ing a dissecting scope and
epifluorescencesource, with light restricted by the micro-scope
aperture. Recent advances (Arrenberget al. 2009) reported zebrafish
lines witheNpHR- enhanced yellow fluorescent protein(eYFP) and
ChR2-eYFP expression controlledby the Gal4/UAS system to allow the
tools to beeasily targeted to specific neuronal subtypes viagenetic
crosses with zebrafish expressing Gal4in various cell populations.
Of note, Arrenberget al. (2009) compared various iterations ofNpHR
and fluorophore and concluded thateNpHR2.0-eYFP had the most
reliable andefficient expression. Photoconverting proteinsKaede and
Dendra were used to approximatethe upper bound of light spread with
lownumerical aperture, small-diameter (50 um)fibers in place of a
microscope aperture, ob-serving that the combination of small-fiber
andneuronal targeting allowed for the stimulationof an
approximately 30-um-diameter spot.
Other groups have reported using Gal4/UAS systems driving
optogenetic tools toexamine cardiac function and
development(Arrenberg et al. 2010), transduction of sensoryneuron
mechanoreception (Low et al. 2010),command of swim behavior
(Arrenberg et al.2009, Douglass et al. 2008), and saccade
genera-tion (Schoonheim et al. 2010). As an alternativeto producing
stable transgenic lines, Zhu et al.(2009) undertook a systematic
examination ofviral infection in zebrafish and found success-ful
ChR2 delivery by the Sindbis and rabiesviruses. Of note, they also
modulated ChR2 ex-pression using a Tet-inducible expression
strat-egy. A specific technical consideration of im-plementing
optogenetic tools in studies usingzebrafish is the stimulation of
neurons thatexpress endogenous light-actived proteins;Arrenberg et
al. (2009) found that 26% of
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control neurons in zebrafish had a firing ratemodulated by
yellow light. This percentage wasreduced to 14% in congenitally
blind zebrafishlines. The remaining response was postulatedto be
due to either expression of other opticallyactivated proteins or a
thermal response, but itwas not investigated further.
MouseBy far, the most widely published optogeneticmodel organism
to date has been the mouse.Mice represent the majority of
transgenic an-imals, including a vast selection of transgeniclines
expressing Cre recombinase in specificsubpopulations of neurons
(Gong et al. 2007).Mouse embryonic stem cells have also
beenamenable to expression and interrogation withoptogenetic
technologies (Stroh et al. 2010).The first report to use
channelrhodopsin inbehaving mammals examined the contributionof
hypothalamic hypocretin (orexin) neuronsto sleep and wakefulness
(Adamantidis et al.2007).
Optogenetic modulation in mouse has alsoyielded control of
monoaminergic systems. Re-cently, in a study that used ChR2,
eNpHR2.0,and TH::Cre transgenic mice to modulatethe locus coeruleus
neurons bidirectionally,these noradrenergic neurons strongly
modu-lated sleep and arousal states (Carter et al.2010). Using the
same TH::Cre transgenicmice, causal relationships were identified
be-tween activity patterns in VTA dopamine neu-rons and reward
behavior in mice, showingthat phasic dopamine release is more
effec-tive than tonic release in driving reward be-havior (Tsai et
al. 2009). Using optogeneticstimulation of axonal terminals in the
nucleusaccumbens, investigators recently discoveredthat dopamine
neurons corelease glutamate(Stuber et al. 2010, Tecuapetla et al.
2010).DAT-Cre mice have been used in conjunc-tion with
Cre-dependent ChR2 to examinemechanisms underlying
dopamine-modulatedaddiction (Brown et al. 2010). And
ChAT-Cretransgenic mice were used in combinationwith Cre-dependent
ChR2 and NpHR viruses
to show that cholinergic interneurons ofthe nucleus accumbens
are key regulators ofmedium spiny neuron activity and can mod-ulate
cocaine-based place preference (Wittenet al. 2010). The
connectivity of striatal mediumspiny neurons themselves has been
describedusing a tetracycline-based ChR2 transgenic sys-tem (Chuhma
et al. 2011).
Direct optogenetic modulation of principaland local-circuit
inhibitory neurons in mousecortex and hippocampus has also
enabledcontributions to understanding the complexityof mammalian
neural circuit dynamics. Reportson the functions of
parvalbumin-expressingfast-spiking interneurons demonstrated
di-rectly their involvement in gamma oscillationsand information
processing in mouse pre-frontal (Sohal et al. 2009) and
somatosensory(Cardin et al. 2009, 2010) cortex. Focal stim-ulation
of pyramidal neurons in Thy1::ChR2mice has enabled rapid,
functional mappingof motor control across the motor cortex(Ayling
et al. 2009, Hira et al. 2009), andaxonal stimulation in regions
contralateral toinjected cortical areas has enabled the mappingof
projection patterns in callosal corticalprojections (Petreanu et
al. 2007). Within localcortical microcircuits, ChR2 has been used
tocharacterize the spatial receptive fields of var-ious neuron
types (Katzel et al. 2010, Petreanuet al. 2009, Wang et al. 2007)
and to study thebasic properties of cortical disynaptic inhibi-tion
(Hull et al. 2009). Optogenetics is alsobeing used to discern the
possible therapeuticmechanism of cortical intervention in
mousemodels of depression (Covington et al. 2010)and to develop
novel strategies for control ofperipheral nerves (Llewellyn et al.
2010).
Mice have also been used to study amyg-dala circuits involved in
fear and anxiety.Johansen and colleagues (2010) used ChR2
todemonstrate the sufficiency of lateral amygdalapyramidal neurons
in auditory cued fearconditioning. In two recent reports,
functionalcircuits within the central amygdala werefurther
delineated, demonstrating that distinctsubpopulations of inhibitory
central amygdalaneurons separately gate the acquisition and
404 Fenno Yizhar Deisseroth
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expression of conditioned fear (Ciocchi et al.2010, Haubensak et
al. 2010). Finally, Tyeand colleagues (2011) described the
differ-ential effects of activating lateral amygdalaprojections
onto central amygdala neurons inregulating anxiety behaviors. These
studiesshed new light on fear and anxiety behaviorsand demonstrate
the utility of optogenetictechniques in dissecting complex local
neuronalcircuits.
RatRats are important for neuroscience researchbecause of their
ability to perform complexbehavioral tasks, the relative simplicity
of theirbrains (compared with human and nonhumanprimates), and the
ability to perform high-density recordings of neural ensembles
duringfree behavior. Recently, virally delivered opto-genetic tools
were used in rats to examine bloodoxygen leveldependent (BOLD)
responsesin functional magnetic resonance imaging(fMRI) (Lee et al.
2010). Driving ChR2 inexcitatory neuronal populations was
sufficientto elicit a BOLD response not only in localcortical
targets (where both the virus and lightdelivery optical fiber were
targeted) but also indownstream thalamic regions, allowing
globalmaps of activity causally driven by defined cellpopulations
to be obtained within intact livingmammals. Optogenetic work in
rats has beenlimited by the availability of viral promotersthat are
capable of driving specific expressionin the absence of transgenic
targeting, but theadvent of transgenic rat lines expressing
Crerecombinase in specific neuronal subtypes (inaddition to
projection targeting) will greatly ex-pand the potential for using
rat models of neuralcircuit function in health and disease.
Outsidethe central nervous system, optogenetic ma-nipulation in
rodents is providing insights intodiverse physiological functions.
ChR2 was usedto modulate rhythmic beating activity in
rodentcardiomyocytes, demonstrating the potentialfor future
applications in this field (Bruegmannet al. 2010), and several
groups have used opto-
genetics to modulate cardiovascular function,breathing, and
blood pressure (Abbott et al.2009a,b; Alilain et al. 2008; Kanbar
et al. 2010)in both anesthetized and awake rats.
PrimateOptogenetic modulation of primate neurons(Han et al.
2009, Diester et al. 2011) has beenexplored by ChR2 delivery to
cortical neuronsof macaques via lentiviral transduction, but
be-havioral responses have not yet been observed.eNpHR2.0 has been
delivered to human neu-ral tissue in the form of ex vivo human
retinasand has shown optogenetic efficacy on physi-ological
measures (Busskamp et al. 2010) withpossible relevance to retinitis
pigmentosa (RP),a disease in which light-sensing cells degener-ate
in the retina. By expressing eNpHR2.0 inlight-insensitive cone
cells, normal phototrans-duction was restored, as well as
center/surroundcomputational features, directional sensitivity,and
light-guided behavior. Additionally, Weicket al. (2010)
demonstrated the functionality ofChR2 in human embryonic stem
cellderivedneurons.
OUTLOOKThe optogenetic toolbox has broadly expandedto include
proteins that are powerful and di-verse in their ionic selectivity,
spectral sen-sitivity, and temporal resolution. Combinedwith
powerful molecular techniques for trans-genic and viral expression
in rodents, zebrafish,and flies, the current generation of
optogenetictools may be adapted to an extensive landscapeof
questions within neuroscience. The currentgeneration of optogenetic
tools has been op-timized for stronger expression, higher
cur-rents, and spectral shifts to allow combinatorialcontrol within
the same volume of space. On-going improvements to the toolbox will
yieldmolecular tools targeted to subcellular com-partments [such as
dendrites or axons (Lewiset al. 2009)], tools for two-photon
activation,and tools that further expand the optical control
www.annualreviews.org The Development and Application of
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of biochemistry. At this moment in time, single-component
optogenetics has become a staple in
neuroscience laboratories, even as many oppor-tunities remain
yet untapped.
DISCLOSURE STATEMENTThe authors are not aware of any
affiliations, memberships, funding, or financial holdings thatmight
be perceived as affecting the objectivity of this review.
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