Download Optogenetics Review1 - Department Of Biological Sciences

The Japanese Society of Developmental Biologists
Develop. Growth Differ. (2013)
doi: 10.1111/dgd.12053
Review Article
Optogenetic manipulation of neural and non-neural
functions
Hiromu Yawo, 1,2,3 * Toshifumi Asano, 1,3,4† Seiichiro Sakai 1,3,4‡ and
Toru Ishizuka 1,3
1
Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, 2-1-1
Katahira, Aoba-ku, Sendai, 980-8577, 2Center for Neuroscience, Tohoku University Graduate School of Medicine,
Sendai 980-8575, 3Japan Science and Technology Agency (JST), Core Research of Evolutional Science & Technology
(CREST), 5 Sanbancho, Chiyoda-ku, Tokyo, 102-0075, 4Japan Society for the Promotion of Science, 5-3-1 Kojimachi,
Chiyoda-ku, Tokyo 102-0083, Japan
Optogenetic manipulation of the neuronal activity enables one to analyze the neuronal network both in vivo and
in vitro with precise spatio-temporal resolution. Channelrhodopsins (ChRs) are light-sensitive cation channels
that depolarize the cell membrane, whereas halorhodopsins and archaerhodopsins are light-sensitive Cl and
H+ transporters, respectively, that hyperpolarize it when exogenously expressed. The cause-effect relationship
between a neuron and its function in the brain is thus bi-directionally investigated with evidence of necessity
and sufficiency. In this review we discuss the potential of optogenetics with a focus on three major requirements
for its application: (i) selection of the light-sensitive proteins optimal for optogenetic investigation, (ii) targeted
expression of these selected proteins in a specific group of neurons, and (iii) targeted irradiation with high spatiotemporal resolution. We also discuss recent progress in the application of optogenetics to studies of nonneural cells such as glial cells, cardiac and skeletal myocytes. In combination with stem cell technology, optogenetics may be key to successful research using embryonic stem cells (ESCs) and induced pluripotent stem cells
(iPSCs) derived from human patients through optical regulation of differentiation-maturation, through optical
manipulation of tissue transplants and, furthermore, through facilitating survival and integration of transplants.
Key words: channelrhodopsin, neuron, optogenetics.
Introduction
Vertebrate and most invertebrate brains consist of
huge number of neurons that are connected to each
other to make a complex network. For example, in a
human brain, there are 1010–12 neurons, each of which
receives 102–3 synapses. The idea that this neuronal
network generates brain function, the mind, via signal
communication was first proposed over 100 years ago
n y Cajal
by a Spanish neuroanatomist, Santiago Ramo
*Author to whom all correspondence should be addressed.
Email: [email protected]
†
Present address: Graduate School of Engineering, Osaka
University, 2-1, Yamadaoka, Suita 565-0871, Japan.
‡
Present address: RIKEN Brain Science Institute, 2-1 Hirosawa,
Wako-shi, Saitama 351-0198, Japan.
Received 17 October 2012; revised 25 February 2013;
accepted 26 February 2013.
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese
Society of Developmental Biologists
(Cajal 1984). To this day our knowledge about the
neuronal network, its organization and function, is still
limited despite extensive research over the past
100 years. Furthermore, how this network is organized
and becomes functional during the development of an
animal remains to be elucidated.
The function of the neural network has been studied
as a cause-effect relationship of stimulation and
response (O’Connor et al. 2009). With the twentiethcentury technological development in electronics, electrophysiology has long been one of the principal
methods for the study of neurons and neural networks.
That is, the neural network is electrically stimulated
and the effects are electrically recorded. For example,
Penfield and his colleagues electrically stimulated various regions of the human brain and electrically
recorded the resulting muscle contractions in their
series of experiments during the 1930s (Penfield &
Rasmussen 1950). With these results they precisely
mapped regions of the cerebral cortex involved in
movement. Nowadays, electrical stimulation is applied
2
H. Yawo et al.
using field or intracellular/patch electrodes. Although
electrical field stimulation is simple, convenient and
has high temporal resolution, the electrical field is generally non-uniform and many untargeted neurons are
stimulated simultaneously. It is thus difficult to identify
which neurons are stimulated. On the other hand, a
single, identified neuron can be selectively stimulated
with an intracellular or whole-cell patch electrode.
However, the number of simultaneous stimulations is
spatially limited as each electrode is independently
manipulated.
Optical stimulation methods have received much
attention recently with the technological development of
modern optics. They have advantages over conventional
electrical stimulation methods: finer spatiotemporal resolution and parallel stimulations at multiple sites (Callaway
€ck 2004). These methods are
& Yuste 2002; Miesenbo
also less harmful and more convenient than electrical
stimulation methods. Another breakthrough combined
optical stimulation with genetic engineering technologies, which is otherwise known as optogenetics. Lightsensing proteins of various living organisms are now
available to be exogenously expressed in neurons and
other target cells both in vivo and in vitro. Cellular functions such as the membrane potential, can thus be
manipulated by light. In this review, we will focus on the
basic principles of optogentic manipulation of neural
and non-neural tissues. Optical probing methods that
use protein sensors have not been discussed in this
review as these investigations have been reviewed
€ck & Kevrekidis 2005; Palmer &
elsewhere (Miesenbo
Tsien 2006; Mank & Griesbeck 2008; Newman et al.
€pfel 2012).
2011; Peterka et al. 2011; Kno
Fundamental molecular biology of
optogenetics
The genes of two channelrhodopsins, channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2), were
first identified in the expressed sequence tag (EST)
project of Chlamydomonas reinhardtii at the Kazusa
DNA Research Institute, Japan (http://www.kazusa.or.
jp/) by three independent groups (Nagel et al. 2002,
2003; Sineshchekov et al. 2002; Suzuki et al. 2003).
The two papers by Nagel et al. (2002, 2003) were
remarkable in that they identified both ChR1 and 2 as
ion channels directly gated by light using Xenopus
oocyte expression system. The existence of these proteins was first proposed following the results from
electrical measurements taken from the intact alga
(Harz & Hegemann 1991; Braun & Hegemann 1999).
Sineshchekov et al. (2002) revealed that both ChR1
and ChR2 (Chlamydomonas sensory rhodopsins A
and B [CSRA and B] in the original paper) mediate the
light-dependent behavior of alga. Suzuki et al. (2003)
showed that ChR1 apoproteins (archaeal-type Chlamydomonas opsin-1 [Acop-1] in the original paper) are
localized in small regions of the plasma membrane
covering the eyespot or stigma, where photoreceptors
had been thought to be concentrated (Melkonian &
Robenek 1980; Kateriya et al. 2004). ChR homologues
have also been identified in other species (Zhang et al.
2011), including, Volvox carteri (VChR1 and VChR2),
Chlamydomonas augustae (CaChR1), Chlamydomonas yellowstonensis (CyChR1), Chlamydomonas
raudensis (CraChR2), Mesostigma viride (MChR1), Dunaliella salina (DChR), and the number of reported
species homologues continues to increase (Ernst et al.
2008; Zhang et al. 2008; Kianianmomeni et al. 2009;
Govorunova et al. 2011; Hou et al. 2012; Watanabe
et al. 2012). Each ChR is a member of the microbialtype (archaeal-type, type I) rhodopsin family with a
core structure of about 300 amino acids. The core
structure consists of seven transmembrane helices
(TM1-7) and a retinal that covalently binds to a consensus Lys residue at the middle of TM7 (Fig. 1A,B).
Light absorption is followed by the photoisomerization
of an all-trans retinal to a 13-cis configuration. This
conformational change allows the channel structure to
become permeable to cations, such as Na+, K+, Ca2+
and H+, (Fig. 1C, Bamann et al. 2008; Ernst et al.
2008; Stehfest & Hegemann 2010). This enables very
rapid (within milliseconds) generation of a photocurrent
across cell membranes expressing ChRs (Nagel et al.
2002, 2003; Boyden et al. 2005; Ishizuka et al. 2006).
Despite extensive studies, researchers have yet to
describe how the cations flow through the molecular
structure (Sugiyama et al. 2009; Ruffert et al. 2011;
Kato et al. 2012; Tanimoto et al. 2013).
When ChR2 is exogenously expressed in neurons,
blue light irradiation evokes an inward current with
membrane depolarization, which opens voltage-gated
sodium channels and calcium channels, and generates
an action potential (Boyden et al. 2005; Li et al. 2005;
Ishizuka et al. 2006). Neuronal activity can also be negatively regulated by light if the neurons are engineered
to express either a Cl-transporting rhodopsin, such
as, NpHR from Natronomonas pharaonis or an H+transporting rhodopsin, such as, archaerhodopsin-3
(Arch) from Halorubrum sodomense and archaerhodopsin-T (ArchT) from Halorubrum strain TP009 (Han &
Boyden 2007; Zhang et al. 2007a, 2011; Chow et al.
2010; Han et al. 2011). The cause-effect relationship
between a neuron and its function in the brain (e.g.,
behavior) is bi-directionally investigated: the necessity
through light-induced silencing of the neuron with
hyperpolarizing rhodopsins and the sufficiency through
light-induced activation with depolarizing rhodopsins.
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
Optogenetic manipulation
Side view
(A)
(B)
N domain
3
Top view
TM3
ECL1
TM2
TM1
N
Extracellular
TM7
TM4
Membrane
All-trans retinal
TM6 ECL3
TM5
ECL2
Intracellular
ICL2
ICL1
C
ICL3
C domain
All-trans retinal
N-
(C)
Basal state
Desensitized photocycle
NOpen state
(O1)
13-cis retinal
Open state
(O2)
Fig. 1. Molecular aspects of channelrhodopsins. (A, B) Crystallographic structure of a chimeric channelrhodopsin, C1C2, which consists
of transmembrane helix (TM)1–5 of ChR1 and TM6 and 7 of ChR2: side view (A) and top view from the extracellular face (B). Although
C1C2 forms a homodimer at N domain, each monomer may form a channel. C, C-terminal; N, N-terminal; ECL1–3, extracellular loops;
ICL1–3, intracellular loops. C. Photocycle of ChR2. A model derived from spectroscopy and photocurrent measurements. For each
state, the number is the wavelength of peak absorbance. With the absorption of blue light, the basal state (P470) is converted to the
open state (O1, P520) via intermediates, P500 and P390. Within a certain dwelling time the 13-cis retinal returns to the all-trans configuration (P470), which closes the gate of an ion channel. However, there is a chance that a molecule may fall into the desensitized photocycle with an open state (O2, P5200 ) of reduced conductance and different ion selectivity (Hegemann et al. 2005; Nikolic et al. 2009;
Berndt et al. 2010). The transition from the desensitized (Des480) to the basal state (D470) is very slow, 10–20 s in the case of ChR2.
The backward transition from P390/3900 or P520/5200 to the basal state (D470/Des480) is facilitated by the absorption of UV or greenyellow light, respectively, and becomes obvious in the case of SFO and SSFO. (A, B), reprinted with modification by permission from
Macmillan Publishers Ltd: Nature (Kato et al. 2012). (C) Reprinted with modification by permission from John Wiley and Sons, Ltd:
Chemphyschem (Stehfest & Hegemann 2010).
To use these optogenetic molecules for neurobiological experiments, we have to meet at least three requirements: (i) selection of the light-sensitive proteins optimal
for optogenetic investigation, (ii) targeted expression of
the above proteins in a specific group of neurons, and (iii)
targeted irradiation with high spatio-temporal resolution.
Molecular optimization
Although ChR2 has been widely used to photostimulate neurons, recent technological developments will
help us exploit the full potential of light-gated ion channels.
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
4
H. Yawo et al.
1. The peak absorption of ChR2 is at 460–470 nm,
the wavelength preferentially absorbed by live tissue
(Yaroslavsky et al. 2002; Aravanis et al. 2007).
Although some native and altered ChRs absorb
green (Yizhar et al. 2011a; Mattis et al. 2012), the
more red-shifted ChRs absorbing red or even near
infra-red light would be more desirable for relatively
deep tissue penetration and/or even irradiation.
Parallel irradiation may also be facilitated by using
ChRs with various wavelength sensitivities in combination with multi-colored optics. Experimentally,
ChR-dependent photostimulation may be used in
combination with fluorescent probes of calcium,
membrane potential or other cellular functions. For
example, the excitation spectrum of Fluo-3, one of
the most popular fluorescent Ca2+ indicators, overlaps with the absorption spectrum of ChR2. Therefore, it would be difficult to measure Fluo-3
fluorescence during ChR2-dependent photostimulation. The irradiation used for the measurement of
Fluo-3 fluorescence would evoke the ChR2 photocurrent, which should inevitably change the intracellular Ca2+ concentration. On the other hand, the
wavelengths 340 and 380 nm, which are optimal
for the use of fura-2, would minimally evoke the
ChR2 photocurrent. Recently, various genetically
encoded fluorescent reporters/sensors have
become available to probe living cells at the func€pfel 2012). ChRs
tional level (Zhao et al. 2011b; Kno
with various absorption spectra would facilitate
these studies.
2. Photocurrents may be enhanced by facilitating protein folding and membrane expression. Mis-folded
or mis-directed molecules are most likely toxic to
(A) Light
the cell through endoplasmic reticulum (ER) stress
(Ron & Walter 2007; Kim et al. 2008). This
becomes apparent when the protein synthesis is
accelerated using powerful promoters such as
those derived from viruses.
3. During blue light irradiation, the ChR2 photocurrent
peaks almost instantaneously, but desensitizes
rapidly to a steady state within several tens of milliseconds (Fig. 2A). It takes several tens of seconds
for full recovery from desensitization. The prominent
desensitization of the ChR2 photocurrent limits its
application for repetitive stimulation at high frequency.
Both the peak and steady-state photocurrents have
their ceilings, even with enhanced light (Fig. 2B) since
desensitization and its rate are enhanced with an
increase in light power density (Fig. 2C).
4. The turning on/off rate of the photocurrent should
be adjusted to the neuron and the stimulation frequency. In the central nervous system (CNS), some
neurons tend to fire at high frequency. The relatively
slow kinetics of ChR2 (τOFF = 10–20 ms) is inadequate to drive these neurons, particularly at high
frequency. Prolonged depolarization often induces
multiple spikes even with irradiation of short duration. ChR variants with small τOFF (Lin et al. 2009;
Wang et al. 2009a; Gunaydin et al. 2010; Wen
et al. 2010; Berndt et al. 2011) would solve these
problems. On the other hand, some variants of
ChRs, such as ChR2(C128S), ChR2(D156A) and
ChR2(C128S/D156A), have very slow deactivation
kinetics and the photocurrent can be terminated
with different colors of light (Berndt et al. 2009; Bamann et al. 2010; Yizhar et al. 2011b; Fig. 1C).
These step-function opsins (SFOs) and stable SFOs
(B)
Current
(C)
Desensitization
0.2 mWmm–2
200 pA
20 ms
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
Fig. 2. Desensitization of ChR2 photocurrent. (A) Photocurrent kinetics of
ChR2. With a pulse irradiation (top), the
photocurrent peaked rapidly and
desensitized to the steady state (bottom). (B) Photocurrent amplitude as a
function of irradiance. Each peak amplitude (r) and steady-state (s) amplitude
were normalized to the peak amplitude
at the maximal irradiance. (C) Desensitization rate as a function of irradiance.
The photocurrent was desensitized
according to a single exponential function with a time constant that is the
reciprocal of the rate constant.
Optogenetic manipulation
(SSFOs) are over two-orders more sensitive to dim
light and are suitable for stimulating relatively deep
neurons.
5. ChRs with variable ion selectivity would enable
broader applications. Although the membrane
potential is shifted to the negative direction in a
light-dependent manner by the use of a Cl-transporting or an H+-transporting protein, such as
NpHR and Arch, its efficiency of this model is limited because only one ion is transported across the
membrane with the absorption of a single photon.
However, if ChRs were designed to be selectively
permeable to Cl or K+, these ChRs would allow
the bulk of ions to flow with the absorption of a single photon. Therefore, their hyperpolarizing effects
would be expected to be much larger than those of
Cl/H+ transporters. ChRs that are not permeable
to Ca2+ are suitable to examine the effects of depolarization per se. On the other hand, those selective
for Ca2+ (Kleinlogel et al. 2011a; Prigge et al. 2012)
could be useful to manipulate the intracellular Ca2+.
Some optimal optogenetic molecules could be
obtained by genome mining (Zhang et al. 2011). The
5
absorption/action spectra were red-shifted in one of
the ChRs from Volvox carteri (VChR1) and in one from
Mesostigma viride (MChR1) (kmax = 520–540 nm).
Variants of ChRs with various properties were also
generated by targeted mutagenesis, by transmembrane helix shuffling, or combinations thereof (Table 1).
Among them, those including TM1-2 of ChR1, generally showed reduced desensitization and enhanced
photocurrents with improved folding/membrane
expression (Lin et al. 2009; Wang et al. 2009a; Lin
2010; Mattis et al. 2012; Prigge et al. 2012). On the
other hand, the photocurrent retardation of ChR1 was
overcome by exchanging TM6 with its counterpart in
ChR2 (Wen et al. 2010). Thus designed channelrhodopsin-green receiver (ChRGR) showed relatively large
photocurrents with red-shifted spectral sensitivity (identical to ChR1), small desensitization and rapid on/off
kinetics. With these advantages, the use of ChRGR
would enable one to inject a current into a neuron with
a time course that could be predicted by the intensity
of light (opto-current clamp). Structural data, such as
that provided by X-ray crystallography, facilitates the
design of ChR variants with the desired spectral sensitivity, ion permeability and so on (Kato et al. 2012).
Table 1. Remarkable channelrhodopsin variants
Remarkable properties
Variants
References
Absorption
Red-shifted ChRs
Expression
Relatively large peak amplitude
Desensitization
Relatively small desensitization
ChRGR†
C1V1‡, C1V1(E162T), C1V1(E122T/E162T)
ChR2(H134R)
ChR2(T159C), ChETATC/ChR2(E123T/T159C)
CatCh/ChR2(L132C)
ChRFR§, ChRWR¶
ChIEF††
C1V1(E162T)
ChR2(H134R)
ChETA/ChR2(E123T/H134R), ChR2(E123A/
T159C), ChR2(H134R/T159C)
CatCh/ChR2(L132C)
ChRWR, ChRFR
ChRGR
ChIEF
C1V1(E162T), C1V1(E122T/E162T)
ChETAA/ChR2(E123A), ChETAT/ChR2(E123T)
ChETATC/ChR2(E123T/T159C)
ChRFR
ChRGR
ChIEF
ChR2(C128T), ChR2(C128A), ChR2(C128S)
ChR2(D156A)
ChR2(C128S/D156A)
CatCh/ChR2(L132C)
CatCh+/ChR2(L132C/T159C)
Wen et al. (2010)
Yizhar et al. (2011b)
Nagel et al. (2005)
Berndt et al. (2011)
Kleinlogel et al. (2011a)
Wang et al. (2009a)
Lin et al. (2009)
Yizhar et al. (2011b)
Nagel et al. (2005)
Berndt et al. (2011)
Speed
Relatively fast kinetics
Bistable (SFO/SSFO)
Ion selectivity
Relatively Ca2+ permeable
Kleinlogel et al. (2011a)
Wang et al. (2009a)
Wen et al. (2010)
Lin et al. (2009)
Yizhar et al. (2011b)
Gunaydin et al. (2010)
Berndt et al. (2011)
Wang et al. (2009a)
Wen et al. (2010)
Lin et al. (2009)
Berndt et al. (2009)
Bamann et al. (2010)
Yizhar et al. (2011b)
Kleinlogel et al. (2011a)
Prigge et al. (2012)
†
ChRGR: ChR-green receiver, ChR1(TM1-5, 7)/ChR2(TM6) chimera. ‡C1V1: ChR1(TM1-2)/VChR1(TM3-7) chimera. §ChRFR: ChR-fast
receiver, ChR1(TM1-2)/ChR2(TM3-7) chimera. ¶ChRWR: ChR-wide receiver, C1C2, ChEF, ChR1(TM1-5)/ChR2(TM6-7) chimera.
††
ChIEF: ChEF(I170V).
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
6
H. Yawo et al.
Targeted expression
Various methods are now available for the targeted
expression of exogenous molecules and have been
extensively reviewed (Yizhar et al. 2011a). For example, virus vectors derived from various serotypes of
adeno-associated virus (AAV), human HIV virus (lentivirus) and Sindbis virus have been made in many laboratories and are distributed worldwide. Alternatively,
various electroporation methods have been devised.
For example, retinal ON bipolar cells were specifically
targeted by introducing ChR2 gene connected to the
mGluR6 promoter sequence through electroporation
(Lagali et al. 2008). The cortical layer-specific expression of ChR2 was induced by timed in utero electroporation of mouse (Petreanu et al. 2007; Hull et al.
2009). In these experiments, the GABAergic interneurons were not usually transfected because they
migrate tangentially from the medial ganglionic eminence (MGE; Nadarajah & Parnavelas 2002). ChRs
can also be efficiently expressed in the spinal cord
motorneurons of the chick embryo (Li et al. 2005; Kastanenka & Landmesser 2010; Sharp & Fromherz 2011)
using in ovo electroporation technology (Odani et al.
2008). A small number of physiologically-identified
neurons could also be targeted for the gene expression with a combination of imaging using single-cell
electroporation methods (Kitamura et al. 2008; Uesaka
et al. 2008; Steinmeyer & Yanik 2012).
Another typical strategy of targeted gene expression
is the generation of transgenic animal models for
experiments (Tables 2,3). Conditional expression systems, such as the Cre/loxP recombinase system, the
tTA-tetO (Tet-On/Off) system and the Gal4/UAS system, are particularly promising. For example, in the
Table 2. Transgenic animal lines for optogenetic manipulations
Animal
Gene structure
References
Mouse,
transgenic
Thy1-ChR2-EYFP
CAG-ChR2-EYFP
OMP-ChR2-EYFP
BAC: VGAT-ChR2-YFP
Arenkiel et al. (2007)
Bruegmann et al. (2010)
Dhawale et al. (2010)
Zhao et al. (2011a)
Halassa et al. (2011)
Zhao et al. (2011a)
Mouse,
knockin
Rat,
transgenic
BAC: ChAT-ChR2(H134R)
-EYFP
BAC: TPH2-ChR2(H134R)
-EYFP
BAC: Pvalb-ChR2(H134R)
-EYFP
BAC: Vglut2-ChR2-YFP
Thy1-NpHR-YFP
Orexin-NpHR
Mrgprd-ChR2(H134R)
-Venus
Thy1-ChR2-Venus
Zhao et al. (2011a)
Zhao et al. (2011a)
€gglund et al. (2010)
Ha
Zhao et al. (2008)
Tsunematsu et al. (2011)
Wang & Zylka (2009b)
Tomita et al. (2009)
Ji et al. (2012)
case of the Cre/loxP mouse system, a driver mouse
retains the cre-recombinase gene from the P2 bacteriophage regulated by a promoter that is predetermined to act in a particular cell type. When it is mated
with mice of another reporter line retaining the gene of
interest in loxP-flanked (“floxed”)-stop or floxed-inverse
(FLEX) cassettes, the productive molecules are
expressed in a particular group of cells in the pups
harboring both the cre-recombinase gene and floxed
gene of interest (Witten et al. 2011; Madisen et al.
2012). The tTA-tetO system has the advantage in that
specific gene expression can be temporally regulated
by the application of a chemical substance, doxycycline (Tanaka et al. 2012). Using this system, ChR2
was selectively expressed in recently activated neurons
in the hippocampus (Liu et al. 2012). Nowadays,
various driver mice have been generated and distributed from researchers and bio-resource facilities
(Madisen et al. 2010; Yizhar et al. 2011a) and the
number of reporter mice for optogenetics is increasing
(Table 3).
The Gal4/UAS system is popular for experiments
using Drosophila and zebrafish. In these animals, very
large numbers of various Gal4-expressing lines have
been produced by chance using enhancer/gene
trapping strategies, and then screened for useful
phenotypes (Scott 2009). Similar variations of geneexpression patterns could be generated by a transgenic system of mammals using a Thy-1.2 genomic
expression cassette that is dependent on differences
in chromosomal integration sites and/or in the number
of inserted copies (Arenkiel et al. 2007; Tomita et al.
2009; Ji et al. 2012). Much progress would be
expected with the generation of reporter lines harboring ChR variants optimized for the various experimental
designs. The Brainbow reporter system, in which the
genes of interest are connected with loxP and its variants in tandem, may be used to express combinations
of optogenetic proteins, which could vary between
neurons (Livet et al. 2007).
Optimizing optical systems
The selection of light sources and light delivery methods has been extensively reviewed (Carter & de Lecea
2011; Yizhar et al. 2011a). Photocurrents generated
by the activation of ChRs are dependent on light
power density (LPD) directed on target cells (Ishizuka
et al. 2006). Although both the peak and the steadystate photocurrents are almost always positively
related to the LPD, those of many ChRs and their variants reach near the maximum with LPD as high as
10 mW/mm2 (Mattis et al. 2012). However, SFOs such
as ChR2 (C128S) and ChR2 (D156A) were found to
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
Optogenetic manipulation
7
Table 3. Reporter animal lines for optogenetic manipulations
Animal, conditional system
Gene structure
References
Mouse, Cre-loxP
Rosa26: floxed-ChR2 (H134R)-tdTomato-WPRE
Rosa26: floxed-ChR2 (H134R)-EYFP-WPRE
Rosa26: floxed-Arch-EGFP-ER2-WPRE
Rosa26: floxed-eNpHR3.0-EYFP-WPRE
Rosa26: CAG-floxed-eNpHR2-EYFP,
Rosa26: CAG-floxed-ChR2(C128A)-mCherry-WPRE
BitetO: ChR2-mCherry, NpHR-EGFP
b-actin: tetO-ChR2(C128S)-EYFP
BitetO: human OPN4 (melanopsin)-mCherry
Rosa26: CAG-FRT-ChR2(C128A)-mCherry-WPRE
Rosa26: CAG-FRT-eNpHR2-EYFP-WPRE
UAS: ChR2(H134R)-mCherry
UAS: NpHR-mCherry
UAS: NpHR-eYFP
UAS: ChRWR-EGFP
UAS: LiGluR
Ptet: ChR2-YFP
UAS: ChR2(H134R)-mCherry
UAS: eNpHR-YFP
UAS: ChR2
UAS: ChR2-YFP
UAS: ChR2-mCherry
Madisen et al. (2012)
Madisen et al. (2012)
Madisen et al. (2012)
Madisen et al. (2012)
Imayoshi et al. (2013)
Imayoshi et al. (2013)
Chuhma et al. (2011)
Tanaka et al. (2012)
Tsunematsu et al. (2013)
Imayoshi et al. (2013)
Imayoshi et al. (2013)
Schoonheim et al. (2010)
Arrenberg et al. (2009)
Arrenberg et al. (2009)
Umeda et al. (2013)
Wyart et al. (2009)
Zhu et al. (2009)
Pulver et al. (2009)
Inada et al. (2011)
Schroll et al. (2006)
Zhang et al. (2007b)
Hwang et al. (2007)
Honjo et al. (2012)
Mouse, tTA-tetO (Tet-On/Off)
Mouse, Flp-FRT
Zebrafish, Gal4-UAS
Zebrafish, itTA-Ptet
Drosophila, Gal4-UAS
be more sensitive to light in the order of 102 or greater
(Berndt et al. 2009; Bamann et al. 2010). This is
because the population light sensitivity of the protein is
dependent on the OFF kinetics as well as dependent
on the protein’s intrinsic light sensitivity (Sugiyama
et al. 2009; Mattis et al. 2012). The SSFO mutant of
ChR2, ChR2 (C128S/D156A) acts as a photon integrator because the peak photocurrent amplitude is
dependent on total photon exposure during irradiation
(Yizhar et al. 2011b). On the other hand, the H+ or Cl
transporters in this model were less sensitive to light,
and completely effective hyperpolarization can only be
induced with a LPD of >10 mW/mm2 (Han & Boyden
2007; Zhang et al. 2007a; Chow et al. 2010; Mattis
et al. 2012). Using whole-cell patch clamping, the
change in a neuronal membrane potential can be
determined by providing a constant or modulated light.
For example, the native response of cells (spontaneous oscillations in the membrane potential, spontaneous impulses, etc.) during given depolarization or
hyperpolarization can be obtained with constant irradiation. Using various protocols, modulated light, such
as square pulses, ramp pulses and sine waves, have
been applied to manipulate the cell (Opto-current
clamp, Yawo 2012). Opto-current-clamp experiments
using Zap-function waves (swept-frequency oscillation)
are important for elucidating the resonance frequency
of a neuron (Gutfreund et al. 1995; Tohidi & Nadim
2009), and with this frequency, the firing pattern of a
local network can be determined (Wen et al. 2010).
Previously, most irradiating devices have delivered
monochromatic light to a single spot. However, it has
become increasingly necessary to develop multicolored and spatiotemporally patterned irradiating
devices. For example, one needs to deliver different
wavelengths to activate one-by-one depolarizing opsins (e.g, ChR2) and hyperpolarizing opsins (e.g,
NpHR; Han & Boyden 2007; Zhang et al. 2009; Chow
et al. 2010; Gradinaru et al. 2010; Zorzos et al. 2010).
Alternative irradiation of blue and yellow light is necessary to exploit the full potential of SFOs and SSFOs
(Berndt et al. 2009; Yizhar et al. 2011b). If a neuron
were designed to express fusion proteins of ChR and
NpHR (Kleinlogel et al. 2011b), it could be manipulated
in either the positive or negative direction, simply by
switching the color of the light between blue and
yellow.
In general, a single neuron receives many convergent inputs from various neurons, each of which fires
with a different pattern, and outputs its activity pattern
divergently as action potentials initiated usually at its
initial segment of the axon. Therefore, to analyze the
input-output relationship of a neuron, a network or
even a brain, systematic experiments using parallel
and spatiotemporally patterned stimulations are necessary. Optogenetics is considered to be the most suit€ck 2004;
able method to achieve this (Miesenbo
Ishizuka et al. 2006). Previously, spatially patterned
photostimulation methods using a laser scanning
microscope have been used for the functional map-
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
8
H. Yawo et al.
ping of the cortex (Wang et al. 2007a; Hira et al.
2009). Laser irradiation was also spatiotemporally controlled using an acousto-optic device (Shoham et al.
2005; Wang et al. 2011). Otherwise, a specimen could
be two-dimensionally targeted on a scanning stage
under a single collimated laser beam (Ayling et al.
2009). In these studies, each targeted area was
sequentially, but not simultaneously, illuminated with
other areas. Recently, two-dimensional array lightemitting diodes (LEDs) have been designed for patterned photostimulation (Grossman et al. 2010). At
present, the irradiance is uneven in any given field on
the specimen because inter-LED space is present.
Nevertheless, this method could become one of the
most ideal tools given that LEDs have high temporal
resolution, high power and multiple colors. Spatial light
modulators based on a digital micro-mirror device
(DMD) or liquid crystal display (LCD) have also been
proposed as other ideal tools for patterned photostimulation (Stirman et al. 2012), since they are currently
used for projecting multi-colored patterned images.
Using DMD, 380 and 505 nm LED lights were alternately applied on a region of interest (ROI) to switch
on and off the light-sensitive ionotropic glutamate
receptor (LiGluR; Wang et al. 2007b). The ChR2expressing neurons were spatially differentiated from
other neurons in the circuit using DMD array in Caenorhabditis elegans (Guo et al. 2009) and in zebrafish
(Zhu et al. 2012). A DMD-based commercial projector
was used in the patterned activation of retinal ganglion
cells that express ChR2 (Farah et al. 2007) and mouse
olfactory bulb in which ChR2 is expressed in the
glomeruli (Dhawale et al. 2010). By using DMD, these
studies could use patterned photostimulation either
temporally or spatially, but not both. Recently, the
image projector was applied to deliver multicolored
light with a pre-programmed spatiotemporal pattern
(Stirman et al. 2012; Sakai et al. 2013) (Fig. 3A,B).
With this projector-managed optical system (PMOS),
the depolarizing rhodopsins (such as, ChR2) and the
hyperpolarizing rhodopsins (such as, ArchT and Mac)
may be differentially activated (Fig. 3C,D). These
devices would facilitate in vitro studies of neuronal networks and their dysfunctions, and studies using small
animal models such as nematode worms, flies and zebrafish. They would also enable one to deliver multicolored and patterned light in the brain of animals in vivo
in combination with a microendoscopy technique (Hayashi et al. 2012; Osanai et al. 2013).
In the future, researchers may be able to devise
light-emitting nanoparticles that could be controlled
and charged by magnetic fields or near-infrared light
to generate specific wavelengths of light (Barandeh
et al. 2012; Yue et al. 2012). If successful, it would
become unnecessary to consider the problem of
inserting many optic fibers into the brain.
Application to non-neural tissues
Although it was originally applied in neural tissues to
manipulate the neuronal activity, optogenetics is widely
applicable for other types of cells and biological systems (Table 4).
Glial cells in the brain tissue may be potential targets
of optogenetic manipulation. For example, astrocytes,
which had been considered merely supportive cells for
neurons, were recently revealed to regulate synaptic
transmission and plasticity and are involved in the regulation of brain function (Volterra & Meldolesi 2005;
Perea et al. 2009; Henneberger et al. 2010; Allaman
et al. 2011; Panatier et al. 2011; Min & Nevian 2012;
Navarrete et al. 2012; Schmitt et al. 2012; Wang et al.
2012), although some controversy remains (Lovatt
et al. 2012). Optogenetic stimulation induced the elevation of cytosolic Ca2+ in ChR2(H134R)-expressing
astrocytes in the brainstem, and triggered respiratory
activity in rats in vivo via an adenosine triphosphate
(ATP)-dependent mechanism (Gourine et al. 2010).
The elevation of cytosolic Ca2+, which triggers the
release of glutamate via anion channels, was also optogenetically induced in astrocytes expressing either
LiGluR, ChR2(H134R) or CatCh (Li et al. 2012). Photostimulation triggered the release of glutamate from
Burgmann glia, which expresses ChR2(C128S), in the
cerebellum (Sasaki et al. 2012). This was followed by
AMPA receptor activation in Purkinje cells and induction of long-term depression of parallel fiber-to-Purkinje
cell synapses via metabotropic glutamate receptor
activation.
In the developing and adult brain, microglia are suggested to reshape synapses and thus to be involved in
the regulation of normal brain function, such as learning and memory as well as pathological reactions
(Nimmerjahn et al. 2005; Wake et al. 2009, 2011;
Tremblay et al. 2010; Paolicelli et al. 2011; Ekdahl
2012; Schafer et al. 2012). Microglia dysfunction is
possibly related to neurodevelopmental disorders such
as obsessive-compulsive disorder and Rett syndrome
(Chen et al. 2010; Derecki et al. 2012). They also give
certain signals to astrocytes through ATP release
(Pascual et al. 2012). Recently, Tanaka et al. (2012)
generated transgenic mice that express ChRs in astrocytes and microglia under the regulation of the tTAtetO system. This transgenic system may facilitate our
understanding of how these glial cells function in the
brain.
Other excitable tissues such as cardiac, skeletal and
smooth muscles are potential targets of optogenetics
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
Optogenetic manipulation
9
Microscope
(A)
DLP® projector
Conjugate plane
Half mirror
DMD
Focusing lens
Filter wheel
Fig. 3. An example of projector-managed optical systems (PMOS). (A) Each
color of light, R, G and B channels,
was patterned by digital micro-mirror
device (DMD) and focused on the
specimen through the microscope and
a multi-bandpass filter, which is made
to pass 430–460, 570–600 and 670–
700 nm. (B) The power of light of the
above PMOS. The relative sensitivities
of ChR2 (r) and ArchT (s) are overlaid.
Note that the band-passed light of
430–460 nm at B-channel would selectively activate ChR2, whereas that of
570–600 nm at G-channel would be
exclusively absorbed by ArchT. (C) A
hippocampal slice of a rat, which
expresses ChR2 (green) ubiquitously in
many neurons under Thy1.2 promotor,
with CA1-regional transfection of ArchT
(red) through AAV vector. Scale, 200
lm. (D) The membrane potential was
recorded from a CA1 pyramidal cell
that expressed both ChR2 and ArchT.
The blue light irradiation at the apical
dendrite evoked action potentials (left),
whereas the yellow light at the somal
region hyperpolarized the membrane
potential (middle). With simultaneous
irradiation of both, the action potential
was no longer evoked (right).
Multi-band-pass filter
Lamp
Objective
lens
(B)
ChR2 ArchT
Specimen
(C)
(D)
20 mV
430–460 nm
(Apical dendrite)
because their functions are dependent on changes in
the membrane potential. Photosensitive cardiomyocytes were differentiated from ChR2(H134R)-expressing embryonic stem cells (ESCs) by optical
modification of their pacemaking activities (Bruegmann
et al. 2010; Abilez et al. 2011). Additionally, transgenic
mice and zebrafish were generated to study arrhythmias and to spatially map the cardiac pace-making
region (Arrenberg et al. 2010; Bruegmann et al. 2010).
In vitro as well as in vivo studies of cardiac dysfunctions will be facilitated by the use of optogenetics
because heart muscles can be directly stimulated
without contact and with high spatiotemporal resolution (Knollmann 2010). A potential clinical application
of optical pacing was suggested by modifying the
580–600 nm
(Soma)
500 ms
pacing of cardiomyocytes through optogenetic stimulation of HEK293 cells, which form syncytia with cardiomyocytes (Jia et al. 2011). Skeletal muscle myotubes
that were developed from ChR2-expressing C2C12
myoblasts, an immortal cell line of murine skeletal
myoblasts originally derived from satellite cells (Yaffe &
Saxel 1977), were demonstrated to be contractile with
optical stimulation, showing twitch-like contractions at
low frequency and tetanus-like contraction at high frequency (Asano et al. 2012). In this study, a line of
C2C12 myoblasts was established to express ChR2
and fused with unmodified C2C12 to form multi-nucleated myotubes (Fig. 4A). The maturation of these photosensitive myotubes was facilitated by rhythmic
stimulation using either an electrical field or blue light,
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
10
H. Yawo et al.
Table 4. Optogenetic manipulation of non-neural tissues
Targeted tissue
Optogenetic molecules
Manipulation
References
Astrocyte
ChR2(H134R)
ATP release
Astrocyte
LiGluR
CatCh
ChR2(C128S)
ChR2(C128S)
ChR2(H134R)
NpHR
ChR2 (transgenic)
ChR2(H134R)
ChR2(H134R)
Glutamate release
Gourine et al. (2010)
Figueiredo et al. (2011)
Li et al. (2012)
Glutamate release, LTD
c-fos induction
Pacemaker function
Sasaki et al. (2012)
Tanaka et al. (2012)
Arrenberg et al. (2010)
Pacemaker function
Bruegmann et al. (2010)
Pacemaker function
Jia et al. (2011)
Pacemaker function
Contraction (twitch and tetanus)
Abilez et al. (2011)
Asano et al. (2012)
Burgmann’s glia
Astrocyte microglia
Zebrafish heart
Mouse ES cell-derived cardiomyocytes
in vivo heart
Canine cardiomyocyte syncytium with
ChR2-expressing HEK cells
Human ES cell-derived cardiomyocytes
Skeletal myotube
ChR2(H134R)
ChR2
Undifferentiated
myoblast
Expression of ChR
iPSC/MSC
Expression of ChR
Differentiation
Multinucleated
myotube
Optogenetic
differentiation/
maturation
Maturation
Contractile muscle
Endodermal
cells
Mesodermal
cells
and they become contractile muscle fibers. To overcome muscle weakness such as muscular dystrophy
and amyotrophic lateral sclerosis (ALS), human muscle
tissue could also be substituted with optogenetically
facilitated myogenic development of myoblasts derived
from induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs) derived from the recipient
(Dezawa 2008). There are also potential bioengineering
applications such as, wireless drive of muscle-powered actuators or microdevices (Feinberg et al. 2007),
as skeletal muscle cells are effective force transducers
that can generate contractile energy efficiently through
biochemical reactions.
Ectodermal
cells
Fig. 4. Optogenetically induced differentiation-maturation. (A) Generation of
photosensitive skeletal muscle from
ChR2-expressing C2C12 myoblasts. (B)
An induced pluripotent stem cell (iPSC)
or a mesenchymal stem cell (MSC)
becomes photosensitive by the introduction of ChR gene. The differentiation
and/or maturation of these cells could
possibly be facilitated by rhythmic photostimulation.
In the above studies, it was noteworthy that undifferentiated cells, such as ESCs and myoblasts, could
have genes of light-sensing proteins to become differentiated photosensitive cells and organs. A significantly
broader application of optogenetics could be facilitated
if used in combination with the ESC/iPSC technology
(Dolmetsch & Geschwind 2011; Dottori et al. 2011;
Shi et al. 2012a). Human cells, particularly cells
derived from patients, could be optogenetically studied
to reveal the mechanisms of dysfunction and to evaluate the effectiveness of treatment (Zhang et al. 2010;
Egawa et al. 2012; Israel et al. 2012; Shi et al.
2012b). This level of research would be further
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
Optogenetic manipulation
facilitated if researchers had open-access to human
cell resources (Wray et al. 2012). ESCs/iPSCs could
be made photosensitive by genetic modification with
ChRs and enabled to differentiate with rhythmic photostimulation, as Ca2+ influx through voltage-dependent
Ca2+ channels is facilitated by depolarization (Fig. 4B).
Alternatively, Ca2+ redistribution could be directly mediated by photo-activated ChRs (Nagel et al. 2003;
Berthold et al. 2008; Caldwell et al. 2008; Ernst et al.
2008; Kleinlogel et al. 2011a; Prigge et al. 2012). Photosensitive differentiated cells could be transplanted
into tissue to study the function of cells or tissue
in vivo or to exogenously regulate function using light
(Weick et al. 2010; Stroh et al. 2011). For example,
iPSC-derived photosensitive dopaminergic neurons
could be optically regulated to release dopamine to
reduce the symptoms of Parkinson’s disease (Wernig
et al. 2008; Gibson et al. 2012). Finally, the optical
drive of photosensitive neurons could facilitate their
survival and integration into the host’s neural network
(Kastanenka & Landmesser 2010; Wyatt et al. 2012)
as these processes are known to be dependent on
the activity in either developing (Hubel et al. 1977;
Harris 1981; Katz 1999; Hensch 2004; Sanes et al.
2012) or adult animals (Koike et al. 1989; van Praag
et al. 1999, 2005; Deisseroth et al. 2004; Komitova
et al. 2006; Tashiro et al. 2007; Waddell & Shors
2008).
11
and Rho-GTPase (Levskaya et al. 2009). Animal rhodopsins have been modified to drive the G-proteincoupled signaling pathway by light (Kim et al. 2005; Li
et al. 2005; Airan et al. 2009; Oh et al. 2010; Ye et al.
2011). It is also possible to manipulate gene expression directly (Takahashi et al. 2007) or indirectly by
light (Ye et al. 2011). Thus, with the development of
sophisticated optic systems, cellular functions may be
elegantly and precisely manipulated.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Mesoscopic Neurocircuitry” (No. 23115501) and Grant-in-Aid for
challenging Exploratory Research (No. 23659105) of
the Ministry of Education, Culture, Sports, Science and
Technology (MEXT) of Japan and the Program for Promotion of Fundamental Studies in Health Sciences of
the National Institute of Biomedical Innovation (NIBIO).
We are grateful to Drs W. Shoji and M. Watanabe for
reviewing the manuscript and to B. Bell for language
assistance.
Author contributions
All authors contributed to writing the paper.
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Optogenetic manipulation has broader applications
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