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St. Petersburg Polytechnical University Journal: Physics and Mathematics 1 (2015) 256–263
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The new method for studying neuronal activity: Optogenetics

Alexander I. Erofeev∗, Maxim V. Matveev, Stanislav G. Terekhin, Olga A. Zakharova,
Polina V. Plotnikova, Olga L. Vlasova
Peter The Great St. Petersburg Polytechnic University, 29 Politechnicheskaya St., St. Petersburg 195251, Russian Federation
Available online 4 December 2015

Abstract
The article is devoted to problems of realization and application of optogenetic methods used to identify reasons of various
diseases, to monitor the biochemical processes of cell activity and to study various organisms. The problems of delivery, embedding
and monitoring the expression of opsin genes into the cell genome of interest have been considered. In the article, the parameters and
properties of various opsins and also the main ways of achievement of precise optical control over cell using opsins were presented.
The rules for choosing the parameters of a light beam and the features of its putting were pointed out. The characteristic properties
of the different measurement technique and recording the experimental quantities were analyzed and given.

Copyright © 2015, St. Petersburg Polytechnic University. Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Optogenetics; Opsin; Photosensitivity; Channelrhodopsin; Halorhodopsin; Lentivirus; Transgenic mouse; Action potential; Fiber.

Introduction Importantly, this method entails creating genetically

Optogenetics is a fundamentally new method of re-
search that has been developed in the last decade in the
Deisseroth Lab of Stanford University (Karl Deisseroth
is a professor of Bioengineering and of Psychiatry and
Behavioral Sciences). Optogenetics involves examining
how cells function by introducing photosensitive compo-
nents into their membranes; these components are capa-
ble of altering the properties of carrier cells in response to
being illuminated by a light beam of certain wavelength
and thus act as the carrier’s fluorescent tags.
∗ Corresponding author.
E-mail addresses: alexandr.erofeew@gmail.com (A. I.
Erofeev), m.v.matveev@bk.ru (M. V. Matveev), stasok32@
yandex.ru (S.G. Terekhin), ozakharpba92@gmail.com (O.
A. Zakharova), plopolina@yandex.ru (P. V. Plotnikova),
olvlasova@yandex.ru (O. L. Vlasova).
coded constructions, i.e. optogenetic instruments which,
upon being delivered to specific cells, change their phys-
iology when exposed to light. These instruments allow
to control by light the electric activity of specific types of
neurons, cell signaling, and other processes. In order to
apply the method it is also necessary to create systems
for delivering light into tissues and for registering the
experimental results. While separate elements of these
methods have existed since the 1970s, they were con-
solidated to create optogenetic methods only in 2005
[1]. Initially, the technology developed was aimed at
neuroscientific research. However, optogenetics turned
out to have a far wider potential. The method allows
to control certain events (with time resolutions on the
order of milliseconds, which corresponds to the dura-
tions of biological processes) in certain types of cells
[2–5].

http://dx.doi.org/10.1016/j.spjpm.2015.12.001
2405-7223/Copyright © 2015, St. Petersburg Polytechnic University. Production and hosting by Elsevier B.V. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
(Peer review under responsibility of St. Petersburg Polytechnic University).
 A. I. Erofeev et al. / St. Petersburg Polytechnical University Journal: Physics and Mathematics 1 (2015) 256–263
This analysis is particularly important since cellular
events must be considered only in the context of other
events occurring in individual tissues and in the body as
a whole.
A brief history of the problem
Francis Crick, the discoverer of the DNA structure,
speculated in 1979 that one of the main problems in the
field of neurosciences is selectively controlling a spe-
cific type of brain cells given that the rest of the cells
remain intact [6]. Since it is impossible to excite a spe-
cific brain area with electrodes with the required preci-
sion, and the effect of various medications is too slow,
Crick concluded that visible light has all the properties
enabling it to be used as an instrument of control. At that
time, however, there were no methods to make specific
cells photosensitive.
Even earlier, in 1971, Stoeckenius and Oesterhelt
showed that bacteriorhodopsin acts as an ion pump
that may be rapidly activated by visible-light photons
[7]. Later other members of this family, halorhodopsin
(1977) and channelrhodopsin (2002), were discovered
[8].
Still, the consensus for a long time had been that this
combination of optical and genetic methods would not
produce the desired effect: firstly, because the foreign
membrane proteins introduced to the cell could be toxic;
secondly, many scientists assumed the light-induced cur-
rents to be too low. Additionally, to absorb photons, bac-
terial opsins need a chemical cofactor, all-trans-retinal.
In the summer of 2005, a study was published demon-
strating it was possible to use a bacterial opsin with-
out adding any other parts, components or reagents [1],
with the neurons rendered photosensitive. In the follow-
ing years, other researchers found that bacteriorhodopsin
and halorhodopsin, as well as channelrhodopsin, are ca-
pable of turning neurons on and off rapidly and without
any risks to cells in response to being irradiated by light
of varying wavelengths. Vertebrate tissues already con-
tain all-trans-retinal, and therefore optogenetic control is
possible in intact brain tissues and even in freely moving
animals.
Modern advances
A number of supremely interesting experiments using
the new technology have been carried out in the recent
seven years. New opsins are under development with the
goal of applying optogenetics to a wide array of stud-
ies on various organisms. For example, in 2008, chan-
nelrhodopsin VChR1 sensitive to yellow light instead
257
of blue was derived from Volvox carteri algae [9]. By
concurrently using several types of channelrhodopsins,
an experimenter may simultaneously control mixed cell
populations: some commands may be given to cells of
the first type by yellow light, and others to the second
type by blue light. The so-called ‘fast’ and ‘slow’ opsins
were also created, allowing to control action potential du-
ration. The former opsins are capable of creating action
potentials up to 200 times per second [10]. Opsins have
already been designed that are sensitive to light whose
frequency is at the boundary between the visible and the
infra-red regions. Waves with this frequency penetrate
the tissues deeper and are more easily focused.
One of the most interesting possibilities of optoge-
netic applications is controlling not only the electrical
events in a neuron but also certain biochemical events.
A lot of medicinal drugs are known to function through
the interaction with the family of membrane receptors
(GPCR). These receptors transduce external signals from
some compound (medication) into cells, thus changing
the intracellular signaling, e.g., the calcium ion levels. If
a photosensitive rhodopsin domain is added to a GPCR,
it is possible to obtain receptors that are sensitive to green
light. These receptors have been termed optoXRs [11].
When a single-component optoXR gene was delivered
via a virus into the brains of laboratory animals, cell-
specific control by light over certain biochemical signal
transmission pathways was successfully exercised [11].
Developing new fiber-optic instruments allowed to
deliver optical beams into any brain area of freely moving
animals. Additionally, a method allowing to simultane-
ously examine optical excitation and recording electrical
impulses was created. Currently it is possible, for exam-
ple, to directly measure the electrical activity in neural
ensembles responsible for motor function while simul-
taneously controlling them through opsins.
The first optogenetic studies on freely moving ani-
mals were aimed at examining the neurons synthesiz-
ing the hypocretin neurotransmitter [12]. These cells are
considered to be responsible for the narcolepsy sleep dis-
order. It was these cells that were discovered to exhibit
specific types of electrical activity leading to awaken-
ing. Optogenetics also helped prove that dopaminergic
neurons are responsible for the sense of joy [13].
Research into the newest methods for treating Parkin-
son’s disease [14,15] has produced some of the best-
known optogenetics experiments. This condition is char-
acterized by impaired transmission of information in the
Substantia nigra pars compacta neurons responsible for
the motor function. Deep brain stimulation has been used
to treat Parkinson’s disease since the 1990s. This proce-
dure involves alternating electrical pulses being sent to
258 A. I. Erofeev et al. / St. Petersburg Polytechnical University Journal: Physics and Mathematics 1 (2015) 256–263
specific brain areas using implanted tools. The potential
efficiency of the treatment strategy is, however, severely
limited, since the electrodes non-selectively stimulate
individual brain cells. A fundamental insight into this
treatment method has been gained through optogenet-
ics. When different types of neurons were activated, un-
expected results were uncovered in parkinsonian mice.
Apparently, the greatest therapeutic effort was achieved
not by stimulating a certain type of cells but by control-
ling the activity of connecting axons.
Genetic methods of opsin gene delivery to specific
neural populations
Opsin genes can be selectively expressed in a spe-
cific pre-chosen type of neurons in the brain. Let us
discuss the main strategies whose efficiency has been
proved to achieve in vivo expression. One of the most
widespread methods of genetic material delivery is using
lentiviruses. Optimal gene expression time is two weeks
after injection. The main advantages of this method are
in a high level of gene expression and in the required ex-
pression persisting for several years. The disadvantages
of using lentiviruses include insufficient specificity and a
low level of expression for several cell-specific promot-
ers. Currently, gene material delivery by lentiviruses is
used in practically all experiments on mammals. Another
common method is delivering the materials by adenoas-
sociated viruses (AAV). The optimal gene expression
time is 3 weeks after injection. A less popular method
is using Cre-dependent AAV expression systems. The
expression time is 3 weeks. The advantages and disad-
vantages of the second and the third methods are similar
to those of the lentiviral method.
Viral systems
In contrast with most of the genetic methods,
lentivirus- and AAV-based viral vectors do not require
using transgenic animal models. These methods allow
obtaining a high opsin gene expression level in both ro-
dents and primates for periods of up to several months.
Lentivirus- and AAV-based vectors used in experiments
have the following parameters:
1. there are over 109 transducing units for lentiviruses;
2. there are over 1012 genome copies for AAV.
Generally, opsin gene expression in the rodent brain
reaches the required level in three weeks after AAV in-
jection and in two weeks after lentivirus injection. Over
six weeks may pass before a stable level of expression
in axonal distal ends is achieved.
Electroporation
A method of in utero electroporation can be used in
certain days of embryonic development. This method can
ensure targeted gene delivery to cortical layers I and III,
and to striatal and hippocampal neurons [16–19]. Unlike
the viral methods, electroporation may deliver DNA of
any size with large promoter segments to achieve high
cellular specificity. Electroporation also allows to inject
numerous gene copies.
Transgenic mice
The required opsin gene expression may be achieved
by using transgene cassettes carrying recombinant pro-
moters, and transgene constructs based on bacterial ar-
tificial chromosomes. Several lines of transgenic mice
expressing ChR2 driven by the Thy1 promoter were gen-
erated without altering their reproductive capacity or any
discernible changes in behavior [20,21].
Cre-dependent expression systems
Even though cell-specific promoters are effective for
attaining the required expression levels in certain types of
neurons, some promoters have weak transcriptional ac-
tivity and it is thus impossible to use them to achieve the
level of expression where opsins may effectively induce
the action potential. Special Cre-dependent AAV-vectors
were created to increase transcriptional activity. These
vectors contain transgene cassettes expressing only in
the presence of Cre in transgenic animal lines. There-
fore, in order to increase expression in a specific type of
cells, an appropriate line of mice must be chosen.
Main strategies of optical control by opsins
Let us discuss the basic experimental methods for
achieving optical control using opsins.
Rapid excitation – channelrhodopsins(ChR)
In some cases the bacterial rhodopsin gene introduced
into neurons may cause a light-induced photocurrent.
Currently, modified opsins are mainly used. In particu-
lar, some codons of algae were replaced by codons of
mammals, which significantly increased the expression
of these genes. Of course, changing codons may cause
 A. I. Erofeev et al. / St. Petersburg Polytechnical University Journal: Physics and Mathematics 1 (2015) 256–263
unforeseen effects, when along with an increased expres-
sion in mammalian neurons, there is a depression of some
other function or a decrease in the expression in other
cell types. For example, introducing the H134R mutant
into ChR2 lead to a twofold increase in photocurrent dur-
ing prolonged stimulation, while the temporal precision
sharply reduced as the channel closing rate decreased
[22].
A significant red shift in the optical absorption spec-
trum of algal protein VChR1 [23] stimulated with yel-
low light (excitation wavelength of 590 nm) without
having any effect on ChR2 (excitation wavelength of
470 nm) permits combined excitation control in vivo.
Most channelrhodopsins studied to date have a rela-
tively low single-channel conductance and broad cation
selectivity. Combined methods, including those using
VCHR1 , allow obtaining various characteristics of cel-
lular photocurrents.
For example, a study of L132Cmutant [24] revealed
that its activation leads to insignificant photocurrents
of calcium ions at physiological concentrations of cal-
cium, and the increase in cytosolic Ca2+ concentration
is mainly engendered by the activation of endogenous
voltage-gated calcium channels due to neuronal mem-
brane depolarization [25]. Second- and even third-order
conductance levels should always be taken into account,
especially when a high conductance of calcium ions is
observed.
Different types of cells, and even different cellular
areas may induce, transport, or respond to increased cal-
cium concentrations in different ways. The latest mod-
eling studies where cellular responses to photostimula-
tion were integrated with the Hodgkin–Huxley model
[26] may be expanded and complemented with second-
order conductances, which means that it is now possible
to predict cellular responses to various photostimulation
methods.
Rapid inhibition – halorhodopsins (NpHR)
Inhibition also plays an important role in optogenetic
studies of the activity of neurons and neural ensembles.
Halorhodopsin (HR) inducing electrogenic Cl− current
is used for these studies, but it typically has a desensitiz-
ing effect. However, a Natronomonas pharaonis homo-
logue gene [32–34] is capable of inducing a stable pho-
tocurrent [35] with a wavelength maximum at 590 nm
(excitation at this wavelength does not elicit any response
from ChR2 , which allows to activate ChR2 and NpHR
independent of bidirectional activity modulation). Un-
like excitatory ChR, NpHR requires constant light ex-
posure. Even though NpHR-assisted inhibition was
259
successfully proved in a model of freely moving worms,
in thin sections from the mammalian brain [35], and also
in a neuronal culture [36], it took a few years to achieve
similar results for intact mammals due to problems with
membrane transport necessitating the use of additional
methods.
Opsin eNpHR2.0 was engineered by various meth-
ods; it is characterized by higher current values [37,21].
This allowed to use it for intact rodent tissues [38], as
well as for primate and human tissues [39]. Ultimately,
eNpHR3.0 was created with even higher photocurrent
values for a moderate light intensity in the yellow range
or red-shifted (up to 680 nm) [26].
It should be noted that it is better to use bidirectional
control for experiments in neuronal function inhibition
and to take into account the stability of the photocur-
rents of inhibitory opsins. Finally, doses of light expo-
sure should be carefully managed, in particular, in order
to prevent tissue overheating during constant exposure.
This is why it is important to control the light intensity
required for inhibition [40].
Step-function opsins (SFO)
Conductance of wild-type ChR2 deactivates after
photostimulation stops (it lasts about 10 ms), while
mutant ChR2 (C128X) (mutations to cysteine-128 and
aspartate-156) deactivates far more slowly [27,28]. For
example, C128T, C128A and C128S-substituted mutant
proteins are characterized by reaction times of 2, 42 and
100 s, respectively [27]. This stable blue light-induced
photocurrent can be stopped by yellow light pulses (560–
590 nm). Mutant genes of this class are called step-
function opsins (SFO), since they allow step control the
membrane potential. This type of control will likelier
lead to an action potential threshold and increase the
probability of endogenous synaptic output [27].
Key differences between SFO and ChR
Let us mention two of these differences. Firstly, the
cellular photosensitivity of SFO is higher than that of
ChR; this is a result of open channel accumulation dur-
ing a light pulse [27,29]. The second difference between
SFO and ChR is driven by an asynchronous nature of
SFO-mediated neuronal activity that does not engage all
expressing neurons into a united pattern caused by pho-
tostimulation.
There currently exist SFOs with deactivation times
up to 30 min [30]; this allows to bring the expressing
neurons to a stable resting potential with subsequent
removal of the light source, i.e. allows to conduct the
260 A. I. Erofeev et al. / St. Petersburg Polytechnical University Journal: Physics and Mathematics 1 (2015) 256–263
subsequent behavioral or physiological experiment with-
out any light or other equipment. Moreover, using pro-
longed low-intensity light pulses removes the hetero-
geneities in the response of the expressing cells. In this
case, even large volumes of tissues may eventually be
driven to saturation levels.
However, despite the wide experimental potential of
SFOs, their use must always be accompanied by addi-
tional electrophysiological studies. This is necessary to
correctly interpret the obtained data.
But even though the above-listed ChRs have many
useful properties, none of them are capable of produc-
ing spike trains with frequencies above 40 Hz, while
many types of neurons and their physiological processes
require high-frequency spike trains (over 40 Hz). Even
with wild-type ChR2 (10 ms), and also H134R (20 ms),
precision control is limited at high frequencies. Substi-
tuting the Glu-123 residue with threonine or alanine lead
to the acceleration of the channel closing kinetics from
10 to 4 ms due to a moderate decrease in photocurrent,
which significantly improved the precision of optoge-
netic control [10]. The E123 mutants are unique among
ChRs as they eliminate the sensitivity of channel ki-
netics to membrane potentials, no matter whether they
act separately or in combination with other H134R and
T159C mutants [10,31]. After these non-linear and non-
stationary effects are removed, it becomes easier to pre-
dict and model the channel response. Opsins of this class
(separate E123 mutants or combinations of other mutants
[10]) are called ChETAs (ChRE123T/A). ChETAs can
be used not only for neurons requiring high-frequency
spike trains, since they help reduce the amount of extra
spikes along with the falsely prolonged depolarization.
ChETAs have been shown to provide enhanced perfor-
mance within intact tissues of mammalian brains [10]; at
the same time, fast deactivation typically leads to a de-
crease in effective cellular sensitivity to prolonged light
pulses, as less channels remain open.
Pharmacological, optogenetic and electric stimulation
These types of stimulation differ from the natural
synaptic transmission on account of the changes in the
conductance of ion flows and membrane potentials. Any
of these stimulation types may affect intracellular mem-
branes, endoplasmic reticula, nuclear complexes, synap-
tic vesicles and mitochondria. These factors need to be
taken into account, especially when studying single neu-
rons. Despite the novelty, the precision and the specificity
of optogenetic methods, their results must be compared
to electric stimulation results under similar conditions.
Even though optogenetics allows to explain exactly how
neurons and neuronal ensembles function, experimen-
tal results, generally, are strongly dependent on types
of neurons and stimulation parameters (frequency, dura-
tion, amplitude, etc.). Choice of opsin (e.g., H134R or
L132C) is also of substantial importance.
A huge variety of genes of microbial opsins are found
in nature, which offers ample opportunities for designing
new optogenetic tools.
Tools for regulating biochemical signaling
The above-described microbial opsin genes (type I)
encode ion channels that control the excitability of neu-
rons by modifying their membrane potential above or
below the action potential generation threshold. While
the advantages of this approach are in its speed and pre-
cision, some experiments require temporal and precise
modulation of intracellular processes.
There is one more type of opsins (type II), for ex-
ample, the photosensitive proteins in mammalian eyes;
these proteins are capable of not only inducing a pho-
tocurrent at light exposure but of acting as G-coupled
protein receptors (GPCRs), and therefore taking part in
intracellular signaling. It is possible to control slow in-
hibition [41] or excitation [42]. Currently a great num-
ber of chimeras [43] between vertebrate rhodopsins and
GPCR families are being developed that may serve as
one-component control tools (among them, dopaminer-
gic, serotonergic and adrenergic receptors which all play
an important role in neurotransmission and neuromodu-
lation). These optogenetic tools are called optoXRs, and
they allow to control intracellular signaling for studying
the behavior of freely moving mice [11].
The speed and precision achieved through methods
of biochemical optogenetics provide opportunities that
are unattainable by pharmacological or genetic methods.
The active development of this area of optogenetics al-
lows to use these technologies for practically all types of
cells.
Selecting light beam parameters and light delivery
After opsin expression was achieved in neurons pre-
senting an interest to researchers, the problem of light
beam delivery arose. The requirements imposed on the
beam’s parameters vary depending on the conditions of
the experiment. For example, beam parameters required
for studying fast oscillations in thin brain slices while
using several opsins in vitro are different from those for
studying the effects of prolonged in vivo stimulation of
certain regions of the animal brain [44]. The parame-
ters of photocurrents induced in neurons by light pulses
 A. I. Erofeev et al. / St. Petersburg Polytechnical University Journal: Physics and Mathematics 1 (2015) 256–263
depend on many factors. Some of them are the type of
expressed opsin, irradiation wavelength, intensity and
duration, and even events that happened before the start
of irradiation. If not all channelrhodopsin molecules re-
cover their initial state after the previous exposure, the
initial response to a light pulse is reduced.
Activation parameters for various opsins
For the ChR2 , ChR2 (H134R), ChR1/2 chimeras,
ChETA (derived from Chlamydomonas reinhardtii)
opsin family, the excitation wavelength is 470 nm, while
the main function of these opsins is cellular membrane
depolarization. This opsin family is capable of fast on/off
switching, which is why these opsins are best suited for
precise activation of neurons. Substituting H134R results
in more photocurrents compared to wild-type ChR2 s. In
particular, the ChR1 /ChR2 chimeras and ChETA produce
spikes up to 200 Hz.
The next group of opsins are the step-functional
opsins (SFOs): ChR2 (C128A), ChR2 (C128S), ChR2
(C128T) (derived from Chlamydomonas reinhardtii).
The excitation wavelength is 470 nm, and a light beam
with a 546-nm wavelength is used for inhibition. The
main function of these opsins is also cellular membrane
depolarization. ChR2 s (with point mutations) are char-
acterized by slow or optically switchable deactivation.
Substituting C128A and C128S result in the longest ac-
tivation and the highest photosensitivity, while C128T
retains a high temporal precision. SFOs can be switched
on and off by blue and green light pulses.
VChR1 (derived from Volvox carteri) has an excita-
tion wavelength of 535 nm, and its main function is also
cellular membrane depolarization. A red shift in the ac-
tivation spectrum (relative to ChR2 ) allows to exercise
combined control.
NpHR, eNpHR (derived from Natronomonas pharao-
nis) have an excitation wavelength of 589 nm, but their
main function is cellular membrane hyperpolarization.
This group of opsins is, in a manner of speaking, a light-
activated pump for chloride ions. These opsins are used
to hyperpolarize neurons with high temporal precision
and stable inhibition over several minutes.
Opto-α1AR, opto-β2AR (obtained synthetically)
have an excitation wavelength of 500 nm; their main
function is biochemical control over the cellular mem-
brane. This group of opsins includes light-activated
GPCRs (activated via the G-protein).
When designing a system of light delivery for opsin
activation, the absorption coefficient for photons with a
certain wavelength must be taken into account. It is pro-
portional to the luminous flux which in turn is the number
261
of photons per surface unit, per time unit. However, an-
other quantity is more convenient to use in experiments;
it is the luminous flux density measured in mW/mm2
and defined as the luminous flux multiplied by a pho-
ton energy. For a wild-type channelrhodopsin ChR2 with
standard levels of expression and light illumination at a
473-nm wavelength, the flux density necessary for initi-
ating the axonal potential is 1–5 mW/mm2 .
As previously noted, the requirements for the duration
of illumination depend on the conditions of various op-
togenetic experiments. In case of optogenetic inhibition
the time range is determined by the duration of this inhi-
bition (flux density of 1–5 mW/mm2 ), while for bistable
optogenetic control a short period of time is required,
and the light beam must have a far lower flux density
(less than 0.01 mW/mm2 ).
For in vitro experiments, when a tissue sample is ob-
served under a microscope, the most appropriate light
sources, i.e., halogen/xenon lamps, LEDs, lasers, can be
placed directly along the microscope beam. For some ex-
periments pulsed illumination is required. Fast shutters
(e.g., Lambda DG-4 or Uniblitzshutter) or pulse lasers
can be used to generate short light pulses. High-power
sources (10–15 mW at the tip of a 100 μm fiber) are
best suited for in vivo studies on freely moving ani-
mals. LEDs can also be used to stimulate cortical layers,
while thin optical fibers must be used to control deeper
brain regions. Optical fibers are used for fabricating the
so-called optrodes which are instruments for simulta-
neously recording electrophysiological parameters and
optically exciting opsins. Fiber thickness is also cho-
sen depending on the nature of the studied object. For
example, for mice with unrestricted movement, the re-
quired thickness is no more than 300 μm, and for rats it is
400 μm. The fact that mammalian brain tissues strongly
absorb light should also be kept in mind when setting
up experiments; for example, light beam intensity at a
500 μm distance from the fiber tip is about 10% of the
initial one.
Currently the Laboratory of Molecular Neurodegen-
eration is developing a hardware and software pack-
age for optogenetic research. The proposed package is
a source of light emission consisting of several bright
LEDs, with special software with the required ex-
perimentally chosen diode parameters for opsins pro-
grammed in.
Methods of recording the experimental data
Various methods of measuring experimental param-
eters are used for optogenetic control. First of all, these
are the methods of obtaining and analyzing images
262 A. I. Erofeev et al. / St. Petersburg Polytechnical University Journal: Physics and Mathematics 1 (2015) 256–263
using various dyes which include Ca2+ -specific dyes
(e.g., fura-3, Fluo-5F), and also voltage-sensitive dyes
(VSDs, e.g., RH-155). Such methods are effective for
measuring the electrical activity in large cell popula-
tions ex vivo and in vivo with a high temporal res-
olution. A two-photon microscope can be used for
imaging with Ca2+ -specific dyes, since noises from
channelrhodopsin photoactivation are virtually absent
during two-photon excitation. Voltage-sensitive dyes are
lipophilic molecules whose optical absorption depends
on membrane potential. Along with high-speed cameras
for recording the changes in optical signal, VSD imaging
is used, which allows to detect the changes in neural elec-
trical activity with high spatial and temporal resolutions
(on the order of μm and ms). The absorption maximum
for the RH-155 dye is 700 μm, while excitation peaks
for opsins are in the 470–590 μm range. Such a differ-
ence in wavelengths allows to simultaneously optically
stimulate opsins and to detect images.
Another class of methods for measuring the pa-
rameters of optogenetic experiments entails simultane-
ous control of animal behavior and electrophysiological
recording. For this goal, special optical fiber-based in-
struments were designed to deliver light beams into the
expression area of opsin genes and for electrophysiolog-
ical measurements.
Conclusion
In this article we detailed the gist of the optogenetic
method, its main components and application areas. This
method is currently being rapidly developed and im-
proved, and applied in an increasing number of scientific
areas. Taking the optogenetic approach to studying vari-
ous neurodegenerative diseases is of the greatest interest
to the authors. For example, in 2013, the abnormalities of
synaptic transmission in a cortical and striatal neuronal
culture were demonstrated on mouse models of Hunting-
ton’s disease using the optogenetic method [45]. Study-
ing this type of problem is the subject of the authors’
current and future research.
Acknowledgment
The authors express their gratitude to I.B. Bezproz-
vanny, the head of the Laboratory of Molecular Neu-
rodegeneration, doctor of biological sciences and Carl
J. and Hortense M. Thomsen Chair in Alzheimer’s Dis-
ease Research, and to the whole team of the laboratory
for consultations and assistance in research.
The part of the study dedicated to staging and testing
optogenetic methods is supported by the Russian Scien-
tific Fund grant no. 14-25-0024.
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