Current Biology 20, R897–R903, October 26, 2010 ª2010 Elsevier Ltd All rights reserved
053
Optogenetic Approaches in Neuroscience
André Fiala1,*, Anna Suska2, and Oliver M. Schlüter2
The recently introduced term ‘optogenetics’ describes
a variety of techniques for expressing genes in nerve cells
that render them responsive to light. This approach makes
use of light-sensitive channel proteins that can be used to
manipulate neuronal function. Using genetic strategies,
these channel proteins can be expressed in neurons
defined by a common genetic identity, which can then be
selectively activated or silenced through illumination.
In this minireview, we shall describe the basic principles
of such manipulative optogenetic approaches in neurosci-
ence and summarize how these tools are being exploited
to investigate neuronal circuits and behavior.
Introduction
The nervous systems of humans and other animals consist of
many diverse types of nerve cells that are interconnected with
each other at synaptic sites, ultimately forming functional
circuits that control behavior. Neuroscientists are united by
the quest to decipher how neuronal circuits cooperatively
function to produce behaviors as a function of some combina-
tion of external stimuli, internal state and prior learned experi-
ence. This task is difficult simply because of the enormous
number and heterogeneity of neurons and their synaptic
connections, which provide a high degree of complexity
even in the brains of invertebrates, and even much more so
in those of higher vertebrates with billions of neurons.
In order to unravel the mode of action of neuronal
networks, a neurobiologist’s dream would be not only to
be able to monitor neuronal activity but also to have control
over distinct sets of neurons and to be able to manipulate
their activity and observe the effect on behavior. This idea
is not new. As the activity of a neuron is based on the depo-
larization of its cell membrane, neuronal activity can be
induced by an experimenter using stimulation electrodes
by which the cell membrane can be artificially depolarized
or hyperpolarized. Although stimulation electrodes have
served, and continue to serve, neuroscientists well for
decades, limitations of this invasive approach are obvious.
One can stimulate either just one cell with a single electrode
inserted into a particular neuron, or a broader area of tissue
in the vicinity of the electrode tip, including all the heteroge-
neous neuron types close to the recording site. Another
disadvantage in larger brains, such as those of mammals,
is that electrophysiological recordings are often performed
in thin slice preparations, excluding the possibility of
following up longer projections interconnecting distant brain
regions. Moreover, keeping an electrode in place in many
cases precludes the simultaneous observation of behavioral
actions of the animal under investigation. The term ‘optoge-
netics’ [1] subsumes the technical approaches that try to
1Georg-August-Universität Göttingen, Johann-Friedrich-Blumenbach-
Institute for Zoology and Anthropology, Molecular Neurobiology of
Behavior, c/o ENI-G, Grisebachstr. 5, 37077 Göttingen, Germany.
2European Neuroscience Institute, Molecular Neurobiology,
Grisebachstr. 5, 37077 Göttingen, Germany.
*E-mail: Andre.Fiala@biologie.uni-goettingen.de
DOI 10.1016/j.cub.2010.08.
Minireview
overcome these obstacles to manipulate widely scattered
but functionally defined groups of neurons.
Optogenetics, as the word indicates, combines two exper-
imental strategies: optical manipulation of neuronal activity
(along with appropriate readouts) and genetic definition of
which neurons are manipulated. The optical part comes
from use of a specific light-sensitive protein expressed in
the neurons of interest. Optical methods are now quite
commonly used for the observation of the structure and func-
tion of neurons. In the simplest case, fluorescent proteins are
used to visualize defined neuronal populations. To observe
neuronal activity, a variety of fluorescence sensor proteins,
indicating those involved in intracellular calcium influx,
synaptic vesicle release or second messenger signaling,
have been designed over the past decade [2,3]. In contrast
to these light-emitting optical tools, the term optogenetics
refers to approaches that exploit channel proteins that have
either been rendered responsive to light or that are inherently
light-sensitive, thereby allowing one to manipulate — that is,
activate or inhibit — neuronal activity [1,3–5].
The genetic part, on the other hand, refers to the fact that
the light-sensitive channels are proteins and can be geneti-
cally encoded. This is of advantage because the identity
and thereby potentially the functional role of a neuron within
a circuit might be determined by its individual pattern of gene
expression. If one finds regulatory elements on the DNA
controlling the expression of genes that are characteristic
for a designated population of neurons, one can specifically
couple the DNA sequence of the light-sensitive protein with
the regulatory element and have it transgenically expressed
in neurons defined by a common genetic identity and,
possibly, common functional role. Several optogenetic
strategies to manipulate neuronal activity with light have
been pursued, all of which have specific advantages and
limitations [1,3–6]. We shall consider those approaches
that have been particularly useful in studying neuronal
circuits and animal behavior, all of which have been concep-
tually important and are technically elegant.
Optogenetic Approaches for Manipulating Neuronal
Activity
One strategy to manipulate neuronal activity by light has
been described by Lima et al. [7] in the fruit fly Drosophila
melanogaster, a model organism that is widely used
because of the sophisticated tools available for restricting
transgene expression to defined neuronal populations.
Using these genetic techniques, Lima et al. [7] expressed
an ion channel protein (P2X2) in neurons that remains closed
until its specific ligand, ATP, binds to it. This genetic
approach is combined with a pharmacological tool,
providing the light-sensitive entity. ATP constrained by
a chemical ‘cage’, a photolabile protecting group, is injected
into the animal and is uncaged by a flash of strong UV light.
The liberated ATP binds to the ATP-dependent channel and
causes cation influx into the neuron, ultimately producing
trains of action potentials. Here, the molecule sensitive to
light is applied ubiquitously throughout the brain, but only
a defined population of neurons expressing P2X2 is able to
respond to the uncaged molecule (Figure 1A).
A second approach relies on the design of chemically
modified ion channel proteins that are rendered
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light-sensitive in their opening or closing [8–12]. To this end, a
photosensitive molecule (‘photoswitch’) is conjugated to the
channel. As one example, Szobota et al. [12] exploited this
concept by chemically modifying an ionotropic glutamate
receptor, an excitatory ion channel naturally present in the
vertebrate brain (Figure 1B). This channel is artificially equip-
ped with a cysteine, serving to connect a glutamate analogue
on a photosensitive arm. The photosensitive chemical is
composed of azobenzene, which switches conformation
upon illumination of light of different wavelengths. Illumina-
tion at lower wavelengths (w380 nm) favors the cis
conformation that bends the arm, allowing binding of the
glutamate analogue to the receptor, ultimately causing the
channel to open. Higher wavelengths (w500 nm) reverse
the conformational change toward the extended trans
Figure 1. Strategies for optogenetic activation
of neurons.
(A) Opening of the ATP-dependent channel
P2X2 is achieved by delivering caged ATP into
the extracellular space and by uncaging the
ATP using a strong flash of UV light. The cation
influx leads to a depolarization of the neuronal
membrane, ultimately causing an excitation of
the neuron expressing the channel protein [7].
(B) A glutamate receptor that is a cation channel
is modified such that its ligand, glutamate, is
tethered to a light-sensitive arm that is attached
to the extracellular part of the channel (LiGluR)
[11]. Illumination with different wavelengths
favors the open or closed conformation, allow-
ing one to depolarize and stop depolarization of
the neuron through different illumination.
(C) The channel protein channelrhodopsin-2
(ChR2) is a monomolecular protein that is in it-
self light sensitive due to a binding site for all-
trans retinal. Illumination of the channel with
blue light causes the opening of the channel,
ultimately leading to a depolarization of the
neuronal membrane [15].
conformation, which extracts the gluta-
mate analogue from the binding pocket
and closes the channel. The useful
feature of this engineered glutamate
channel (LiGluR) is that it allows for
bidirectional control of the channel state
by two different wavelengths: the
channel, and thereby the neuron’s
activity, can be precisely turned on and
off. This very efficient tool has the limita-
tion that the chemical compound
tethered to the designed glutamate
channel has to be added to cells or, in
the case of animals, injected. It has,
however, been successfully used in
zebrafish larvae, where the animals can
simply be bathed in a solution containing
the photosensitive compound [12,13].
In recent years, a variety of different
channel proteins have been engineered
using this principle of chemically tagging
a channel protein with a light-sensitive
group that influences open and closed
states, for example, the synthetic
azobenzene-regulated K+ channel (SPARK) [8], or a mutated
variant of the light-regulated K+ channel with reversed
polarity that allows light-induced depolarization (D-SPARK)
[9]. It is likely that, given the large variety of diverse channels
present in neurons and the ability to chemically engineer light
switches, a valuable array of optogenetic tools will be
created in the future that may offer specific advantages
with respect to illumination wavelengths, ion conductance
and temporal precision in switching channels on and off.
A third strategy relies on the characterization of the ‘chan-
nelrhodopsins’ (ChR1, ChR2, VChR1) isolated from the green
algae Chlamydomonas reinhardtii [14,15] and Volvox carteri
[16]. The most widely used channelrhodopsin, ChR2, is a
rather non-specific, single component cation channel that
has a binding moiety for all-trans retinal, a chemical
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compound that renders the channel sensitive to blue light. If
expressed in a neuron, it incorporates into the cell
membrane. In the presence of all-trans retinal, which is natu-
rally abundant in vertebrate neurons (but has to be supplied
in the food in the case of Drosophila or the nematode Caeno-
rhabditis elegans), illumination causes the channel to open,
ultimately resulting in depolarization of the respective
neurons (Figure 1C). One advantage of this protein is its
very rapid opening and closing, which can be controlled by
short light flashes in the range of milliseconds. This is useful,
as the natural signaling unit of neurons, the action potential,
is a millisecond-lasting depolarization of the membrane.
Theoretically, one can therefore determine the exact firing
pattern of the neuron by applying light stimuli at a millisecond
time scale [6]. ChR2 has been successfully used in various
transgenic animals, such as C. elegans [17–22], Drosophila
[23–26], zebrafish [27] and mice (for example [28–30]). As
a proof of principle, it has even been expressed in the frontal
cortex of rhesus monkeys infected with a viral vector ex-
pressing ChR2 DNA [31].
Shortly after the development of ChR2 as a neurobiological
tool, an inhibitory counterpart, the chloride pump ‘halorho-
dopsin’ (NpHR), isolated from the bacteria Natronomonas
pharaoni, was described [32,33]. The light-induced chloride
influx hyperpolarizes neuronal membranes, so that action
potential generation can be suppressed and neurons
silenced. It is fortunate that the activation spectrum of
NpHR is in the yellow range and complementary to that of
ChR2, which is most efficiently activated by blue light.
By expressing both proteins in the same cell, one can either
activate or silence it by illumination with different wave-
lengths [32,33].
Since the first description of ChR2 and NpHR as neurobi-
ological tools, considerable efforts have been made to
change or improve their biophysical properties, for example,
by mutating the amino-acid sequence of ChR2 [34], by
searching for new ChR2 variants in other algae that are
activated by different wavelengths [16], or by adding intra-
cellular trafficking signals that improve the subcellular local-
ization of the channel protein at the plasma membrane
[35,36]. As alternative tools silencing neurons, light-driven
proton pumps from several bacteria have been described
[37,38], underlining that nature might provide an arsenal of
proteins that are useful for optogenetic approaches still wait-
ing to be discovered. In the following, we will describe how
far these tools have been used to investigate neuronal
circuits underlying behavior.
Optogenetic Analysis of Neuronal Circuits
To understand how neuronal circuits function we need to
know how they are constructed, a difficult problem given
the huge numbers of neurons that constitute many brains,
particularly of vertebrates. Neuronal activity is usually re-
corded in isolated tissue preparations or in vivo. Despite
the precise resolution of the activity pattern recorded with
electrodes, the specific connections between neurons and/
or the source that triggers a particular neuron under investi-
gation are usually elusive. A conventional approach to
resolve this problem and to determine whether or not one
neuron synapses with another is to stimulate the first neuron
and record activity from the second, potential target cell.
This approach is suitable for determining local connections
of neurons, for example in slice preparations, often the
preferred preparation for electrophysiological analysis in
vertebrate brains. But this approach has two limitations: first,
it is useful only for investigating the interactions of a limited
number of neurons; and second, long-range connections,
especially in vertebrate brains, are usually cut during slicing.
Optogenetic tools offer new ways of overcoming these
limitations. First, the optical trigger does not interfere with
the electrical recordings. With classical electrodes for stimu-
lating axons, the investigator has to rely on the temporal
delay between the synaptic response and the electrical
stimulation ‘artefact’. With high-frequency stimulations, the
artefacts of the stimulator and the recording electrode over-
lap and compromise the data analysis. Second, the input
axons can be selectively targeted by cell- or region-specific
expression of the optogenetic tools. As the axons survive the
slice preparation, the long-range connections remain and
can be specifically activated by light (Figure 2).
In an elegant study [39], selective expression of ChR2 in
neurons projecting onto the dendritic tree of pyramidal cells
in the mouse barrel cortex was used to map the subcellular
organization of synaptic connections. To increase the spatial
resolution, action potential generation was blocked and
synaptic release triggered, presumably by calcium influx
through the activated ChR2. The dendritic tree was then
scanned with a cylindrical laser beam with a diameter of
w10 mm. The axons of thalamic nuclei, the whisker motor
cortex and local excitatory pyramidal neurons impinged
onto distinct single domains of the recorded pyramidal cells,
indicating a strict topographic organization of the synaptic
connections in neocortical neurons. In another approach,
ChR2 expression in the layer-5 motor cortex of transgenic
mice enabled the topographic mapping of motor cortex
neurons with muscle innervations [40]. Although this had
already been done with classical electrodes, resulting in
the ‘homunculus’ for motor-cortical representations, the
light-based mapping technique might in the future allow for
the automated investigation of changes during skilled
training or of nervous system damage.
One promising way of using ChR2 in neuronal circuit
function analysis relies on the combination of inducible
optogenetic tools with the increasing number of Cre recom-
binase driver mouse lines [41]. This technology enables
cell-type restricted manipulation of neuronal circuits, and
has recently been used to analyze the neuronal origins of
broad oscillations of neuronal activity that are considered
to clock information processing in the brain. To analyze
the role of different neuronal populations in generating
gamma oscillations in the neocortex, Cardin et al. [42]
used a combination of Cre-inducible ChR2 with parvalbu-
min-positive interneuron-specific or pyramidal cell-specific
Cre driver mouse lines [42]. Light-driven activation of par-
valbumin neurons with varied frequencies selectively ampli-
fied gamma oscillations in the barrel cortex. The modulation
was specific for the parvalbumin fast-spiking interneurons,
as the same activation in pyramidal cells only amplified
lower frequency oscillations. These data support the role
of parvalbumin fast-spiking interneurons in the generation
of gamma oscillations in the cortex. The authors further
tested how sensory inputs are dependent on the gamma
cycle. Whisker evoked action potentials were recorded in
the barrel cortex. The timing of the sensory input was varied
relative to a gamma cycle elicited with light. The amplitude
and precision of the sensory response were maximally
close to the peak of the gamma oscillations, indicating a
causal dependence of information processing on brain
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oscillations. This study nicely demonstrates how optoge-
netic tools can be used to dissect the network function of
individual neuronal populations and their contribution to
information processing.
Optogenetic Activation of Reflexive Behaviors
One aim of neuroscience is to reveal how behavior is gener-
ated and controlled by neuronal circuits integrating sensory
stimuli, internal states and prior experience. Observing
neuronal activity can, of course, help to clarify which neurons
are actually involved in certain types of behavior, for example
how sensory modalities are encoded or which neurons are
active during behavioral actions. This approach is purely
correlative, however, and determining causal connections
between neuronal activity and behavior requires manipula-
tive techniques. The inactivation or silencing of defined
neuronal elements within a neuronal circuit can reveal which
neurons are necessary for a specific behavioral task. And
activating neurons artificially, for example optogenetically
using light, should make it possible to determine which
neurons are sufficient to elicit specific behaviors. The idea
of inducing behavior optogenetically has been first tested
in simple types of behavior and in small animals with
a manageable neuronal circuitry.
The first experimental demonstration that animal behavior
can be optogenetically controlled was done with C. elegans
[15]. When ChR2 was expressed in muscle cells of the body
wall, illumination caused strong contractions of the worm.
But when ChR2 was expressed in mechanosensory neurons,
light caused a withdrawal movement that is typically evoked
by mechanical stimulation [17]. Conversely, illuminating
animals that expressed the chloride pump NpHR in body
wall muscles or motor neurons caused paralysis [32]. These
experiments were conceptually important as they provided
a first proof that behavior can indeed be optogenetically
controlled in living, behaving animals if the genetic targeting
of the channel is precise.
If one tries to understand reflexive behaviors as responses
to external stimuli, one initial question would be which
Figure 2. A strategy for a specific activation of
long-range connections in slice preparations.
(A) Glutamatergic afferents that originate in
hippocampus project, among other structures,
to the nucleus accumbens (NAc). Electrophys-
iological analysis of this pathway in NAc
requires a specific stimulation of those axons.
This is achieved by injecting a ChR2-encoding
virus into the hippocampus of a living animal.
(B) After several days during which the ChR2
is expressed in hippocampal neurons, slices
of the NAc are prepared. (C) Upon light stimula-
tion of the NAc, only the axons originating in
the hippocampus will be activated, and (D) a
postsynaptic response of a NAc neuron can
be recorded by a patch pipette.
sensory neurons are sufficient to elicit
a particular more or less complex motor
response. This can be tested optoge-
netically by circumventing the sensory
stimulus and activating sensory cells
through light. The Drosophila larva
provides a model organism that is
particularly well suited for optogenetic approaches, given
the techniques available for expressing transgenes in very
distinct neuronal populations, and because fly larvae are
relatively transparent [23–26]. In this animal, an escape
response characterized by a typical rolling behavior could
be induced by light-activation of a distinct class of mechano-
sensory neurons. This escape response is observed under
natural conditions if the larvae are attacked by parasitoid
wasps [26]. In adult Drosophila, an aversive locomotion
response to CO2 could be mimicked by light-activating
sensory neurons that are specifically responsive to CO2
[24]. Similarly, activation of sugar-sensitive gustatory neu-
rons triggers the reflexive extension of the fly’s ‘tongue’,
the proboscis [25].
These examples illustrate how, in invertebrates, selective
activation of sensory neurons can induce a specific behavior.
Similar approaches have now also been applied to the more
complex nervous systems of vertebrates. In zebrafish
embryos, a well established model system in which trans-
genes can be expressed in defined neuronal subsets, a
reflexive escape movement could be induced by ChR2-
mediated light-activation of a certain class of somatosen-
sory neurons [27].
As elegant as these studies producing ‘sensory illusions’
by activating distinct sensory neurons are, they do not reveal
how relatively complex behavioral actions are actually
induced and maintained. The behavioral repertoire of
animals and humans consists to a large degree of diverse
motor patterns, which often require the precise interplay of
antagonistic sets of muscles. Of course, the initiation and
control of defined motor patterns is accomplished by
neuronal circuits. Here, the relatively high stereotypy and
rhythmicity of many movements has led to the concept of
central pattern generators, groups of neurons that are inter-
connected in such a way that a rhythmic pattern of activity is
produced and conveyed via motor neurons to the appro-
priate set of muscles. In several cases, it has been possible
to optogenetically activate those circuits that induce or
maintain the behavioral action.
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In fruit flies, Lima et al. [7] were able to show that light-acti-
vated neurons located within a reflex circuit do indeed
induce a coordinated escape response — the animals
started flying away. Similarly, a pattern generator evoking
courtship song behavior in Drosophila was optogenetically
identified [43]. In zebrafish larvae, the light-gated channel
LiGluR was expressed in subsets of neurons within the spinal
chord [12]. Activating these cells caused an oscillatory
movement of the fish tail, resembling the natural swimming
behavior of the larval fish. Conversely, the expression of
tetanus toxin, which prevents synaptic transmission from
these neurons, reduced swimming behavior. Clearly, these
neurons are not only necessary, but also sufficient to activate
a central pattern generator that makes the animals swim
forward.
Optogenetic Induction of Behavioral Plasticity
Animals produce more than just reflexive movements. A key
feature of virtually all nervous systems is their ability to
change, a property underlying experience-dependent
behavioral plasticity — learning and memory. Obviously,
any understanding of how neuronal circuits function must
include knowledge about how they are modulated. Optoge-
netic approaches can contribute significantly to this quest.
The first approach to induce learning by optogenetically
activating modulatory neurons has been made in Drosophila
larvae [23]. The larvae of fruit flies readily associate an odor
stimulus with a rewarding sugar stimulus, ultimately
rendering the odor stimulus more attractive. Conversely,
they can associate an odor with a highly concentrated salt
stimulus, which induces a more aversive memory for the
odor. When ChR2 was expressed specifically in dopami-
nergic neurons, and those neurons were activated through
light while the animals smell a certain odor, an aversive
memory for that odor was formed [23]. Conversely, when
tyraminergic and octopaminergic neurons were activated
through ChR2, a simultaneously perceived odor was learned
to be more attractive [23]. These experiments show that
memory formation can be triggered using an optogenetic
technique. Recently, the behavior-reinforcing properties of
dopaminergic neurons for aversive learning were demon-
strated optogenetically in adult flies as well [44], in this
case using the light-induced release of caged ATP in combi-
nation with the P2X2 receptor.
In mammals, the situation is similar in that the transmitter
dopamine has been implicated in mediating the reinforcing
effects of salient stimuli during learning, though in this case
in the context of rewarding signals. In a fascinating study,
Tsai et al. [29] demonstrated that light-activating dopami-
nergic neurons in mice can indeed cause reward learning.
By specifically targeting ChR2 to dopaminergic neurons,
they were able to induce precise firing patterns in these cells
by illumination. To do so, they inserted an optical fiber into
the mouse brain with the tip ending close to the ventral
tegmental area, a brain region where dopaminergic neurons
involved in reward learning are located. In a behavioral para-
digm, the mice were placed in a chamber with characteristic
color and texture of the floor; simultaneously, the mice were
subject to phasic optogenetic activation of dopaminergic
neurons. After one day, the mice were placed in a second,
differently outfitted chamber, and dopaminergic neurons
were only tonically light-activated at a low frequency. Inter-
estingly, in a choice situation the animals showed a learned
preference for the chamber paired with the phasic activation
of dopaminergic neurons [29]. These experiments nicely
demonstrate the behavior-reinforcing and rewarding proper-
ties of dopaminergic neurons. The virtue of this approach is
that the animal can freely behave during the experiment, and
neuronal activity can still be induced specifically, because
the actuator protein responsive to light is genetically
targeted.
This is only one side of the coin, however: obviously, it
would be of interest to know what effects on their target
structures are caused by the release of modulatory transmit-
ters. Neuromodulatory transmitters, such as dopamine,
often exert their effects via the activation of metabotropic,
G-protein-coupled receptors (GPCRs). These, in turn, affect
second-messenger pathways that modulate neuronal excit-
ability or transmitter release. In order to optically activate
these intracellular signaling cascades, Airan et al. [45] have
developed an ingenious technique: they designed chimaeric
transmembrane proteins with extracellular parts derived
from the light-sensitive rhodopsin, and intracellular parts
derived from GPCRs, specifically various adrenergic recep-
tors. In cells expressing these ‘optoXRs’, distinct second-
messenger cascades could be induced through illumination.
In a behavioral experiment, optoXRs were expressed in
neurons of the nucleus accumbens, a brain region involved
in reward learning and a target of dopaminergic neurons of
the ventral tegmental area. When the optoXR construct
carrying the intracellular domains of a b2-adrenergic
receptor was used, the mice developed a significant place
preference memory for the chamber associated with the
optogenetical induction of the intracellular signaling
cascade. These results show that not only can neuronal
excitation be induced optogenetically, but the effect of
intracellular, modulatory signaling cascades can be
mimicked through illumination.
Outlook
In this minireview we have been able to cover only some
aspects of a rapidly developing field. Most likely, new opto-
genetic channel proteins will be created in the upcoming
years that will outperform the first generation of tools. But
already one can see what properties these new tools should
have. First, the currents conducted by the channel should be
sufficiently high for an efficient depolarization or hyperpolar-
ization of neurons. In this respect, ChR2 still suffers from
poor conductivity. Those approaches that incorporate
chemical modifications — for example, uncaging of ligands
such as ATP — have clear advantages in this respect.
It would seem to be desirable to have single-component
proteins that provide similarly high conductance. Stronger
ion fluxes could theoretically be achieved by longer open
probabilities of the channels at the cost of temporal preci-
sion. Furthermore, ion selectivity could be improved — the
microbial opsin channel ChR2 conducts monovalent ions,
such as Na+, K+ and H+, but also Ca2+. The Na+ conductance
particularly contributes to the depolarization of the neuron,
which can then trigger action potentials by the neuronal
machinery; however, the additional Ca2+ conductance
complicates the use of the ChR2. Ca2+ can directly trigger
second messenger pathways or synaptic transmission,
hence compromising the results, when ChR2 is used solely
for the purpose for depolarizing the neuronal membrane.
Second, the wavelength used for opening/closing the
optogenetic channel is of importance. It would be advanta-
gous to be able to use wavelengths that do not overlap
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with the excitation wavelengths of fluorescent sensor
proteins that could be used for simultaneous optical imaging
of neuronal activity. Attempts to improve the biophysical
properties of respective channel proteins are already
ongoing [46].
As fascinating as the development of optogenetic tech-
niques is, the question may be asked how far we can really
get by analyzing neuronal circuits underlying behavior using
optogenetic tools. In principle, the precision of which
neurons are activated relies on how precisely a gene expres-
sion pattern can be used to target the channel protein. If
sufficiently specific expression can be achieved, for example
through the combination of neuronal silencers and activa-
tors, a precise determination of whether particular neurons
are required and/or sufficient for a certain behavior can be
obtained. Additional information from sensors to register
the natural activation pattern during a behavior can unam-
biguously identify the role of genetically identifiable neurons
in a behavior. As outlined above, for invertebrates, the
connection between behavior and a neuronal subtype has
been determined in some case. But these examples primarily
involve the analysis of modulatory neurons and some highly
specified sensory neurons.
A related question that is more relevant for complex brains
is how well gene expression patterns reflect specific
neuronal functions. Dissecting the role of neurons in more
complex networks, such as the cortex of a vertebrate brain,
will depend on the identification of neurons that function in
specific behaviors and a unique genetic identity to target
the optogenetic tools into those neurons. Specifically acti-
vated genes in subpopulations of neurons could potentially
be hitchhiked using genetic switches, such as the Cre/loxP
system, and be used to activate the optogenetic tools in
those neurons. This approach might add to the toolbox opto-
geneticists can use to dissect the function of individual or
small populations of neurons in certain behaviors, even in
more complex brains as those of vertebrates.
The advent of optogenetic tools in neuroscience has been
received with great hope, not least due to the prospect of
being able to better investigate and understand the neuronal
mechanisms underlying neurological disorders. In general,
fundamental research in analyzing the causes of neurological
disorders has already profited from optogenetic techniques,
for example in the context of analyzing cellular sources of Par-
kinson’s disease [47], for counteracting epileptiform activity
[48] or for restoring visual perception in mice that are blind
due to photoreceptor degeneration (reviewed in [49]). At the
moment, it cannot be foreseen if and when optogenetics
may be used to treat patients. The limiting factor here is the
safe delivery of transgenes into somatic cells of humans. If
this hurdle can be overcome in the future, optogenetics might
provide a useful strategy for many medical applications.
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