Optogenetics is a biological technique to control the activity of
neurons
A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. N ...
or other cell types with light. This is achieved by
expression
Expression may refer to:
Linguistics
* Expression (linguistics), a word, phrase, or sentence
* Fixed expression, a form of words with a specific meaning
* Idiom, a type of fixed expression
* Metaphorical expression, a particular word, phrase, o ...
of
light-sensitive ion channels,
pumps
A pump is a device that moves fluids (liquids or gases), or sometimes slurries, by mechanical action, typically converted from electrical energy into hydraulic energy. Pumps can be classified into three major groups according to the method they u ...
or
enzymes
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
specifically in the target cells. On the level of individual
cells,
light-activated enzymes and
transcription factor
In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fu ...
s allow precise control of biochemical signaling pathways.
In
systems neuroscience, the ability to control the activity of a genetically defined set of neurons has been used to understand their contribution to decision making, learning, fear memory, mating, addiction, feeding, and locomotion. In a first medical application of optogenetic technology, vision was partially restored in a blind patient.
Optogenetic techniques have also been introduced to map the
functional connectivity
Resting state fMRI (rs-fMRI or R-fMRI) is a method of functional magnetic resonance imaging (fMRI) that is used in brain mapping to evaluate regional interactions that occur in a resting or task-negative state, when an explicit task is not being ...
of the brain''.'' By altering the activity of genetically labelled neurons with light and using imaging and electrophysiology techniques to record the activity of other cells, researchers can identify the
statistical dependencies between cells and brain regions.
In a broader sense, optogenetics also includes methods to
record cellular activity with
genetically encoded indicators.
In 2010, optogenetics was chosen as the "Method of the Year" across all fields of science and engineering by the interdisciplinary research journal ''
Nature Methods
''Nature Methods'' is a monthly peer-reviewed scientific journal covering new scientific techniques. It was established in 2004 and is published by Springer Nature under the Nature Portfolio. Like other ''Nature'' journals, there is no external edi ...
''. At the same time, optogenetics was highlighted in the article on "Breakthroughs of the Decade" in the academic research journal ''
Science
Science is a systematic endeavor that builds and organizes knowledge in the form of testable explanations and predictions about the universe.
Science may be as old as the human species, and some of the earliest archeological evidence for ...
''.
History
In 1979,
Francis Crick
Francis Harry Compton Crick (8 June 1916 – 28 July 2004) was an English molecular biologist, biophysicist, and neuroscientist. He, James Watson, Rosalind Franklin, and Maurice Wilkins played crucial roles in deciphering the helical struc ...
suggested that controlling all cells of one type in the brain, while leaving the others more or less unaltered, is a real challenge for neuroscience.
Francis Crick
Francis Harry Compton Crick (8 June 1916 – 28 July 2004) was an English molecular biologist, biophysicist, and neuroscientist. He, James Watson, Rosalind Franklin, and Maurice Wilkins played crucial roles in deciphering the helical struc ...
speculated that a technology using light might be useful to control neuronal activity with temporal and spatial precision but at the time there was no technique to make neurons responsive to light.
By early 1990s LC Katz and E Callaway had shown that light could uncage glutamate.
Heberle and Büldt in 1994 had already shown functional heterologous expression of a bacteriorhodopsin for light-activated ion flow in yeast. Later in 1995,
Georg Nagel
Georg Nagel (born 24 August 1953 in Weingarten, Germany) is a biophysicist and professor at the Department for Neurophysiology at the University of Würzburg in Germany. His research is focused on microbial photoreceptors and the development of op ...
et al. and
Ernst Bamberg
Ernst Bamberg (b. in Krefeld) is a German biophysicist and director emeritus of the Department of Biophysical Chemistry at the Max Planck Institute of Biophysics.
Career
Bamberg received his PhD in physical chemistry from the University of Ba ...
tried the heterologous expression of microbial rhodopsins (also bacteriorhodopsin and also in a non-neural system, Xenopus oocytes) (Nagel et al., 1995, FEBS Lett.) and showed light-induced current.
The earliest genetically targeted method that used light to control rhodopsin-sensitized neurons was reported in January 2002, by
Boris Zemelman and
Gero Miesenböck
Gero Andreas Miesenböck (born 15 July 1965) is an Austrian scientist. He is currently Waynflete Professor of Physiology and Director of the Centre for Neural Circuits and Behaviour (CNCB) at the University of Oxford and a fellow of Magdalen ...
, who employed ''
Drosophila
''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
''
rhodopsin
Rhodopsin, also known as visual purple, is a protein encoded by the RHO gene and a G-protein-coupled receptor (GPCR). It is the opsin of the rod cells in the retina and a light-sensitive receptor protein that triggers visual phototransduction ...
cultured mammalian neurons.
In 2003, Zemelman and Miesenböck developed a second method for light-dependent activation of neurons in which single ionotropic channels TRPV1, TRPM8 and P2X2 were gated by photocaged ligands in response to light.
Beginning in 2004, the Kramer and Isacoff groups developed organic photoswitches or "reversibly caged" compounds in collaboration with the
Trauner group that could interact with genetically introduced ion channels. TRPV1 methodology, albeit without the illumination trigger, was subsequently used by several laboratories to alter feeding, locomotion and behavioral resilience in laboratory animals. However, light-based approaches for altering neuronal activity were not applied outside the original laboratories, likely because the easier to employ channelrhodopsin was cloned soon thereafter.
Peter Hegemann
Peter Hegemann (born 11 December 1954) is a Hertie Senior Research Chair for Neurosciences and a Professor of Experimental Biophysics at the Department of Biology, Faculty of Life Sciences, Humboldt University of Berlin, Germany. He is known ...
, studying the
light response of green algae at the University of Regensburg, had discovered photocurrents that were too fast to be explained by the classic g-protein-coupled
animal rhodopsins. Teaming up with the electrophysiologist Georg Nagel at the Max Planck Institute in Frankfurt, they could demonstrate that a single gene from the alga ''
Chlamydomonas
''Chlamydomonas'' is a genus of green algae consisting of about 150 speciesSmith, G.M. 1955 ''Cryptogamic Botany Volume 1. Algae and Fungi'' McGraw-Hill Book Company Inc of unicellular flagellates, found in stagnant water and on damp soil, ...
'' produced large photocurrents when expressed in the oocyte of a frog. To identify expressing cells, they replaced the cytoplasmic tail of the algal protein with a fluorescent protein
YFP, generating the first generally applicable optogenetic tool.
They stated in the 2003 paper that "expression of ChR2 in oocytes or mammalian cells may be used as a powerful tool to increase cytoplasmic Ca
2+ concentration or to depolarize the cell membrane, simply by illumination".
Karl Deisseroth
Karl Alexander Deisseroth (born November 18, 1971) is an American scientist. He is the D.H. Chen Professor of Bioengineering and of psychiatry and behavioral sciences at Stanford University.
He is known for creating and developing the technolo ...
in the Bioengineering Department at Stanford published the notebook pages from early July 2004 of his initial experiment showing light activation of neurons expressing a channelrhodopsin.
In August 2005, his laboratory staff, including graduate students
Ed Boyden
Edward S. Boyden is an American neuroscientist at MIT. He is the Y. Eva Tan Professor in Neurotechnology, a faculty member in the MIT Media Lab and an associate member of the McGovern Institute for Brain Research. In 2018 he was named a Howard Hu ...
and
Feng Zhang
Feng Zhang (; born October 22, 1981) is a Chinese-American biochemist. Zhang currently holds the James and Patricia Poitras Professorship in Neuroscience at the McGovern Institute for Brain Research and in the departments of Brain and Cognitive ...
, in collaboration with Georg Nagel, published the first demonstration of a single-component optogenetic system, in neurons
using the channelrhodopsin-2(H134R)-eYFP construct from Nagel and Hegemann.
Zhuo-Hua Pan
Zhuo-Hua Pan (; born 1956) is a Chinese-American neuroscientist, known for his foundational contributions to optogenetics. He is the Edward T. and Ellen K. Dryer Endowed Professor of Ophthalmology at Wayne State University, and Scientific Directo ...
of
Wayne State University
Wayne State University (WSU) is a public research university in Detroit, Michigan. It is Michigan's third-largest university. Founded in 1868, Wayne State consists of 13 schools and colleges offering approximately 350 programs to nearly 25,000 ...
, researching on restore sight to blindness, tried channelrhodopsin out in ganglion cells—the neurons in our eyes that connect directly to the brain. Pan's first observation of optical activation of retinal neurons with channelrhodopsin was in August 2004 according to Pan, a month after Deisseroth's initial observation. Indeed, the transfected neurons became electrically active in response to light, and in 2005 Zhuo-Hua Pan reported successful in-vivo transfection of channelrhodopsin in retinal ganglion cells of mice, and electrical responses to photostimulation in retinal slice culture.
This approach was eventually realized in a human patient by
Botond Roska
Botond Roska (born 1969) is a Hungarian medical doctor and biomedical researcher. Much of his research is on the pathways of visual perception and how to treat diseases that cause blindness.
Early life and education
Botond Roska was born in 1969 ...
and coworkers in 2021.
In April 2005,
Susana Lima and Miesenböck reported the first use of genetically-targeted P2X2
photostimulation Photostimulation is the use of light to artificially activate biological compounds, cells, tissues, or even whole organisms. Photostimulation can be used to noninvasively probe various relationships between different biological processes, using on ...
to control the behaviour of an animal.
They showed that photostimulation of genetically circumscribed groups of neurons, such as those of the
dopaminergic
Dopaminergic means "related to dopamine" (literally, "working on dopamine"), dopamine being a common neurotransmitter. Dopaminergic substances or actions increase dopamine-related activity in the brain. Dopaminergic brain pathways facilitate d ...
system, elicited characteristic behavioural changes in fruit flies.
In October 2005, Lynn Landmesser and Stefan Herlitze also published the use of channelrohodpsin-2 to control neuronal activity in cultured hippocampal neurons and chicken spinal cord circuits in intact developing embryos.
In addition, they introduced for the first time vertebrate rhodopsin, a light-activated G protein coupled receptor, as a tool to inhibit neuronal activity via the recruitment of intracellular signaling pathways also in hippocampal neurons and the intact developing chicken embryo.
The groups of
Alexander Gottschalk and Georg Nagel made the first ChR2 mutant (H134R) and were first to use channelrhodopsin-2 for controlling neuronal activity in an intact animal, showing that motor patterns in the roundworm ''
C. elegans
''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (r ...
'' could be evoked by light stimulation of genetically selected neural circuits (published in December 2005). In mice, controlled expression of optogenetic tools is often achieved with cell-type-specific Cre/loxP methods developed for neuroscience by
Joe Z. Tsien Joe Z. Tsien is a neuroscientist who pioneered Cre/lox-neurogenetics in the mid-1990s, a versatile toolbox for neuroscientists to study the complex relationships between genes, neural circuits, and behaviors. He is also known as the creator of the s ...
back in the 1990s to activate or inhibit specific brain regions and cell-types ''in vivo''.
In 2007, the labs of Boyden and Deisseroth (together with the groups of Gottschalk and Nagel) simultaneously reported successful optogenetic inhibition of activity in neurons.
In 2007, Nagel and Hegemann's groups started the optogenetic manipulation of cAMP. In 2014, Avelar et al. reported the first rhodopsin-guanylyl cyclase gene from fungus. In 2015, Scheib et al. and Gao et al. characterized the activity of the rhodopsin-guanylyl cyclase gene. And Shiqiang Gao et al. and Georg Nagel, Alexander Gottschalk identified it as the first 8 TM enzyme rhodopsin.
Awards
The powerful impact of optogenetic technology on brain research has been recognized by numerous awards to key players in the field.
In 2010, Georg Nagel, Peter Hegemann and Ernst Bamberg were awarded the
Wiley Prize in Biomedical Sciences and they were also among those awarded the Karl Heinz Beckurts Prize in 2010. In the same year, Karl Deisseroth was awarded the inaugural
HFSP Nakasone Award for "his pioneering work on the development of optogenetic methods for studying the function of neuronal networks underlying behavior".
In 2012, Bamberg, Deisseroth, Hegemann and Nagel were awarded the Zülch Prize by the
Max Planck Society
The Max Planck Society for the Advancement of Science (german: Max-Planck-Gesellschaft zur Förderung der Wissenschaften e. V.; abbreviated MPG) is a formally independent non-governmental and non-profit association of German research institutes. ...
, and Miesenböck was awarded the Baillet Latour Health Prize for "having pioneered optogenetic approaches to manipulate neuronal activity and to control animal behaviour."
In 2013, Nagel and Hegemann were among those awarded the
Louis-Jeantet Prize for Medicine
Established in 1986, the Louis-Jeantet Prizes are funded by the ''Fondation Louis-Jeantet'' and awarded each year to experienced researchers who have distinguished themselves in the field of biomedical research in one of the member states of t ...
. Also that year, year Bamberg, Boyden, Deisseroth, Hegemann, Miesenböck and Nagel were jointly awarded
The Brain Prize
The Brain Prize, formerly known as The Grete Lundbeck European Brain Research Prize, is an international scientific award honouring "one or more scientists who have distinguished themselves by an outstanding contribution to neuroscience and who are ...
for "their invention and refinement of optogenetics."
In 2017, Deisseroth was awarded the
Else Kröner Fresenius Research Prize for "his discoveries in optogenetics and hydrogel-tissue chemistry, as well as his research into the neural circuit basis of depression."
In 2018, the
Inamori Foundation presented Deisseroth with the
Kyoto Prize
The is Japan's highest private award for lifetime achievement in the arts and sciences. It is given not only to those that are top representatives of their own respective fields, but to "those who have contributed significantly to the scientific, ...
for "spearheading optogenetics” and "revolutionizing systems neuroscience research."
In 2019, Bamberg, Boyden, Deisseroth, Hegemann, Miesenböck and Nagel were awarded the
Rumford Prize
Founded in 1796, the Rumford Prize, awarded by the American Academy of Arts and Sciences, is one of the oldest scientific prizes in the United States. The prize recognizes contributions by scientists to the fields of heat and light. These terms ...
by the
American Academy of Arts and Sciences
The American Academy of Arts and Sciences (abbreviation: AAA&S) is one of the oldest learned societies in the United States. It was founded in 1780 during the American Revolution by John Adams, John Hancock, James Bowdoin, Andrew Oliver, and ...
in recognition of "their extraordinary contributions related to the invention and refinement of optogenetics."
In 2020, Deisseroth was awarded the
Heineken Prize for Medicine from the
Royal Netherlands Academy of Arts and Sciences
The Royal Netherlands Academy of Arts and Sciences ( nl, Koninklijke Nederlandse Akademie van Wetenschappen, abbreviated: KNAW) is an organization dedicated to the advancement of science and literature in the Netherlands. The academy is housed ...
, for developing optogenetics and hydrogel-tissue chemistry. On the same year, Miesenböck, Hegemann and Nagel jointly received the
Shaw Prize in Life Science and Medicine.
In 2021, Hegemann, Deisseroth and
Dieter Oesterhelt received the
Albert Lasker Award for Basic Medical Research
The Albert Lasker Award for Basic Medical Research is one of the prizes awarded by the Lasker Foundation for a fundamental discovery that opens up a new area of biomedical science. The award frequently precedes a Nobel Prize in Medicine; almost 5 ...
.
Description
![Example of optogenetic activation of prefrontal cortex](https://upload.wikimedia.org/wikipedia/commons/1/15/Example_of_optogenetic_activation_of_prefrontal_cortex.jpg)
Optogenetics provides millisecond-scale temporal precision which allows the experimenter to keep pace with fast biological information processing (for example, in probing the causal role of specific
action potential
An action potential occurs when the membrane potential of a specific cell location rapidly rises and falls. This depolarization then causes adjacent locations to similarly depolarize. Action potentials occur in several types of animal cells, ...
patterns in defined neurons). Indeed, to probe the neural code, optogenetics by definition must operate on the millisecond timescale to allow addition or deletion of precise activity patterns within specific cells in the brains of intact animals, including mammals (see Figure 1). By comparison, the temporal precision of traditional genetic manipulations (employed to probe the causal role of specific genes within cells, via "loss-of-function" or "gain of function" changes in these genes) is rather slow, from hours or days to months. It is important to also have fast readouts in optogenetics that can keep pace with the optical control. This can be done with electrical recordings ("optrodes") or with reporter proteins that are
biosensor
A biosensor is an analytical device, used for the detection of a chemical substance, that combines a biological component with a physicochemical detector.
The ''sensitive biological element'', e.g. tissue, microorganisms, organelles, cell rece ...
s, where scientists have fused fluorescent proteins to detector proteins. Additionally, beyond its scientific impact optogenetics represents an important case study in the value of both
ecological conservation (as many of the key tools of optogenetics arise from microbial organisms occupying specialized environmental niches), and in the importance of pure basic science as these
opsin
Animal opsins are G-protein-coupled receptors and a group of proteins made light-sensitive via a chromophore, typically retinal. When bound to retinal, opsins become Retinylidene proteins, but are usually still called opsins regardless. Most pro ...
s were studied over decades for their own sake by biophysicists and microbiologists, without involving consideration of their potential value in delivering insights into neuroscience and neuropsychiatric disease.
Light-activated proteins: channels, pumps and enzymes
The hallmark of optogenetics therefore is introduction of fast light-activated channels, pumps, and enzymes that allow temporally precise manipulation of electrical and biochemical events while maintaining cell-type resolution through the use of specific targeting mechanisms. Among the microbial opsins which can be used to investigate the function of neural systems are the
channelrhodopsin
Channelrhodopsins are a subfamily of retinylidene proteins ( rhodopsins) that function as light-gated ion channels. They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis: movement in response to light. Express ...
s (ChR2, ChR1, VChR1, and SFOs) to excite neurons and
anion-conducting channelrhodopsin
Anion-conducting channelrhodopsins are light-gated ion channels that open in response to light and let negatively charged ions (such as chloride) enter a cell. All channelrhodopsins use retinal as light-sensitive pigment, but they differ in their ...
s for light-induced inhibition. Indirectly light-controlled
potassium channel
Potassium channels are the most widely distributed type of ion channel found in virtually all organisms. They form potassium-selective pores that span cell membranes. Potassium channels are found in most cell types and control a wide variety of cel ...
s have recently been engineered to prevent action potential generation in neurons during blue light illumination. Light-driven ion pumps are also used to inhibit neuronal activity, e.g.
halorhodopsin Halorhodopsin is a light-gated ion pump, specific for chloride ions, found in archaea, known as halobacteria. It is a seven-transmembrane retinylidene protein from microbial rhodopsin family. It is similar in tertiary structure (but not primary seq ...
(NpHR),
enhanced halorhodopsins (eNpHR2.0 and eNpHR3.0, see Figure 2),
archaerhodopsin
Archaerhodopsin proteins are a family of retinal-containing photoreceptors found in the archaea genera '' Halobacterium'' and ''Halorubrum''. Like the homologous bacteriorhodopsin (bR) protein, archaerhodopsins harvest energy from sunlight to pum ...
(Arch), fungal opsins (Mac) and enhanced bacteriorhodopsin (eBR).
Optogenetic control of well-defined biochemical events within behaving mammals is now also possible. Building on prior work fusing vertebrate
opsins
Animal opsins are G-protein-coupled receptors and a group of proteins made light-sensitive via a chromophore, typically retinal. When bound to retinal, opsins become Retinylidene proteins, but are usually still called opsins regardless. Most p ...
to specific
G-protein coupled receptors
G protein-coupled receptors (GPCRs), also known as seven-(pass)-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors (GPLR), form a large group of protein family, evolution ...
a family of
chimeric single-component optogenetic tools was created that allowed researchers to manipulate within behaving mammals the concentration of defined intracellular messengers such as cAMP and IP3 in targeted cells.
Other biochemical approaches to optogenetics (crucially, with tools that displayed low activity in the dark) followed soon thereafter, when optical control over small GTPases and adenylyl cyclase was achieved in cultured cells using novel strategies from several different laboratories.
Photoactivated adenylyl cyclase
Photoactivated adenylyl cyclase (PAC) is a protein consisting of an adenylyl cyclase Protein domain, enzyme domain directly linked to a BLUF domain, BLUF (blue light receptor using FAD) type light sensor domain. When illuminated with blue light, th ...
s have been discovered in fungi and successfully used to control cAMP levels in mammalian neurons. This emerging repertoire of optogenetic actuators now allows cell-type-specific and temporally precise control of multiple axes of cellular function within intact animals.
Hardware for light application
Another necessary factor is hardware (e.g. integrated fiberoptic and solid-state light sources) to allow specific cell types, even deep within the brain, to be controlled in freely behaving animals. Most commonly, the latter is now achieved using the fiberoptic-coupled diode technology introduced in 2007,
though to avoid use of implanted electrodes, researchers have engineered ways to inscribe a "window" made of zirconia that has been modified to be transparent and implanted in mice skulls, to allow optical waves to penetrate more deeply to stimulate or inhibit individual neurons.
[ • Explained by ] To stimulate superficial brain areas such as the cerebral cortex, optical fibers or
LED
A light-emitting diode (LED) is a semiconductor Electronics, device that Light#Light sources, emits light when Electric current, current flows through it. Electrons in the semiconductor recombine with electron holes, releasing energy i ...
s can be directly mounted to the skull of the animal. More deeply implanted optical fibers have been used to deliver light to deeper brain areas.
Complementary to fiber-tethered approaches, completely wireless techniques have been developed utilizing wirelessly delivered power to headborne LEDs for unhindered study of complex behaviors in freely behaving organisms.
Expression of optogenetic actuators
Optogenetics also necessarily includes the development of genetic targeting strategies such as cell-specific promoters or other customized conditionally-active viruses, to deliver the light-sensitive probes to specific populations of neurons in the brain of living animals (e.g. worms, fruit flies, mice, rats, and monkeys). In invertebrates such as worms and fruit flies some amount of
all-trans-retinal
Retinal (also known as retinaldehyde) is a polyene chromophore. Retinal, bound to proteins called opsins, is the chemical basis of visual phototransduction, the light-detection stage of visual perception (vision).
Some microorganisms use retin ...
(ATR) is supplemented with food. A key advantage of microbial opsins as noted above is that they are fully functional without the addition of exogenous co-factors in vertebrates.
Technique
![Optogenetic stimulation consists of several steps](https://upload.wikimedia.org/wikipedia/commons/2/28/Optogenetic_stimulation_consists_of_several_steps.png)
The technique of using optogenetics is flexible and adaptable to the experimenter's needs. Cation-selective channelrhodopsins (e.g. ChR2) are used to excite neurons, anion-conducting channelrhodopsins (e.g. GtACR2) inhibit neuronal activity. Combining these tools into a single construct (e.g. BiPOLES) allows for both inhibition and excitation, depending on the wavelength of illumination.
Introducing the microbial opsin into a specific subset of cells is challenging. A popular approach is to introduce an engineered viral vector that contains the optogenetic actuator gene attached to a specific
promoter such as
CAMKIIα, which is active in excitatory neurons. This allows for some level of specificity, preventing e.g. expression in
glia
Glia, also called glial cells (gliocytes) or neuroglia, are non-neuronal cells in the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses. They maintain homeostasis, form mye ...
cells.
A more specific approach is based on transgenic "driver" mice which express
Cre recombinase
Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme uses a topoisomerase I-like mechanism to carry out site specific recombination events. The enzyme (38kDa) is a member of the integrase family of site ...
, an enzyme that catalyzes recombination between two lox-P sites, in a specific subset of cells, e.g.
parvalbumin
Parvalbumin (PV) is a calcium-binding protein with low molecular weight (typically 9-11 kDa). In humans, it is encoded by the ''PVALB'' gene. It is not a member of the albumin family; it is named for its size (''parv-'', from Latin ''parvus'' smal ...
-expressing
interneurons
Interneurons (also called internuncial neurons, relay neurons, association neurons, connector neurons, intermediate neurons or local circuit neurons) are neurons that connect two brain regions, i.e. not direct motor neurons or sensory neurons. In ...
. By introducing an engineered viral vector containing the optogenetic actuator gene in between two lox-P sites, only the cells producing Cre recombinase will express the microbial opsin. This technique has allowed for multiple modified optogenetic actuators to be used without the need to create a whole line of transgenic animals every time a new microbial opsin is needed.
After the introduction and expression of the microbial opsin, a computer-controlled light source has to be optically coupled to the brain region in question.
Light-emitting diode
A light-emitting diode (LED) is a semiconductor device that emits light when current flows through it. Electrons in the semiconductor recombine with electron holes, releasing energy in the form of photons. The color of the light (cor ...
s (LEDs) or fiber-coupled
diode-pumped solid-state lasers (DPSS) are frequently used. Recent advances include the advent of wireless head-mounted devices that apply LEDs to the targeted areas and as a result, give the animals more freedom to move.
Fiber
Fiber or fibre (from la, fibra, links=no) is a natural or artificial substance that is significantly longer than it is wide. Fibers are often used in the manufacture of other materials. The strongest engineering materials often incorporate ...
-based approaches can also be used to combine optical stimulation and
calcium imaging
Calcium imaging is a microscopy technique to optically measure the calcium (Ca2+) status of an isolated cell, tissue or medium. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca2+ ions ...
.
This enables researchers to visualize and manipulate the activity of single neurons in awake behaving animals. It is also possible to record from multiple deep brain regions at the same using
GRIN lenses connected via optical fiber to an externally positioned photodetector and photostimulator.
Technical challenges
Selective expression
One of the main problems of optogenetics is that not all the cells in question may express the microbial opsin gene at the same level. Thus, even illumination with a defined light intensity will have variable effects on individual cells. Optogenetic stimulation of neurons in the brain is even less controlled as the light intensity drops exponentially from the light source (e.g. implanted optical fiber).
It remains difficult to target opsin to defined subcellular compartments, e.g. the plasma membrane, synaptic vesicles, or mitochondria.
Restricting the opsin to specific regions of the plasma membrane such as
dendrite
Dendrites (from Greek δένδρον ''déndron'', "tree"), also dendrons, are branched protoplasmic extensions of a nerve cell that propagate the electrochemical stimulation received from other neural cells to the cell body, or soma, of the n ...
s,
somata
The soma (pl. ''somata'' or ''somas''), perikaryon (pl. ''perikarya''), neurocyton, or cell body is the bulbous, non-process portion of a neuron or other brain cell type, containing the cell nucleus. The word 'soma' comes from the Greek '' σῶ ...
or
axon terminal
Axon terminals (also called synaptic boutons, terminal boutons, or end-feet) are distal terminations of the telodendria (branches) of an axon. An axon, also called a nerve fiber, is a long, slender projection of a nerve cell, or neuron, that condu ...
s provides a more robust understanding of neuronal circuitry.
Mathematical modelling shows that selective expression of opsin in specific cell types can dramatically alter the dynamical behavior of the neural circuitry. In particular, optogenetic stimulation that preferentially targets inhibitory cells can transform the excitability of the neural tissue, affecting non-transfected neurons as well.
Kinetics and synchronization
The original channelrhodopsin-2 was slower closing than typical cation channels of cortical neurons, leading to prolonged depolarization and calcium influx. Many channelrhodopsin variants with more favorable kinetics have since been engineered.
">556]
A difference between natural spike patterns and optogenetic activation is that pulsed light stimulation produces synchronous activation of expressing neurons, which removes the possibility of sequential activity in the stimulated population. Therefore, it is difficult to understand how the cells in the population affected communicate with one another or how their phasic properties of activation relate to circuit function.
Optogenetic activation has been combined with
functional magnetic resonance imaging
Functional magnetic resonance imaging or functional MRI (fMRI) measures brain activity by detecting changes associated with blood flow. This technique relies on the fact that cerebral blood flow and neuronal activation are coupled. When an area o ...
(ofMRI) to elucidate the
connectome
A connectome () is a comprehensive map of neural connections in the brain, and may be thought of as its "wiring diagram". An organism's nervous system is made up of neurons which communicate through synapses. A connectome is constructed by tr ...
, a thorough map of the brain's neural connections.
Precisely timed optogenetic activation is used to calibrate the delayed hemodynamic signal (
BOLD
In typography, emphasis is the strengthening of words in a text with a font in a different style from the rest of the text, to highlight them. It is the equivalent of prosody stress in speech.
Methods and use
The most common methods in W ...
) fMRI is based on.
Light absorption spectrum
The opsin proteins currently in use have absorption peaks across the visual spectrum, but remain considerably sensitive to blue light.
This spectral overlap makes it very difficult to combine opsin activation with genetically encoded indicators (
GEVIs,
GECIs
Genpact is an American professional services firm legally domiciled in Bermuda with its headquarters in New York City, New York. The company currently employs more than 100,000 people and provides services to clients in over 30 countries worldwi ...
,
GluSnFR,
synapto-pHluorin Synapto-pHluorin is a genetically encoded optical indicator of vesicle release and recycling. It is used in neuroscience to study transmitter release. It consists of a pH-sensitive form of green fluorescent protein (GFP) fused to the luminal side of ...
), most of which need blue light excitation. Opsins with infrared activation would, at a standard irradiance value, increase light penetration and augment resolution through reduction of light scattering.
Spatial response
Due to scattering, a narrow light beam to stimulate neurons in a patch of neural tissue can evoke a response profile that is much broader than the stimulation beam.
In this case, neurons may be activated (or inhibited) unintentionally. Computational simulation tools
are used to estimate the volume of stimulated tissue for different wavelengths of light.
Applications
The field of optogenetics has furthered the fundamental scientific understanding of how specific cell types contribute to the function of biological tissues such as neural circuits ''in vivo''. On the clinical side, optogenetics-driven research has led to insights into
Parkinson's disease
Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms usually emerge slowly, and as the disease worsens, non-motor symptoms becom ...
and other neurological and psychiatric disorders such as
autism
The autism spectrum, often referred to as just autism or in the context of a professional diagnosis autism spectrum disorder (ASD) or autism spectrum condition (ASC), is a neurodevelopmental condition (or conditions) characterized by difficulti ...
,
Schizophrenia
Schizophrenia is a mental disorder characterized by continuous or relapsing episodes of psychosis. Major symptoms include hallucinations (typically hearing voices), delusions, and disorganized thinking. Other symptoms include social withdra ...
,
drug abuse
Substance abuse, also known as drug abuse, is the use of a drug in amounts or by methods which are harmful to the individual or others. It is a form of substance-related disorder. Differing definitions of drug abuse are used in public health, ...
, anxiety, and
depression.
An experimental treatment for blindness involves a channel rhodopsin expressed in
ganglion cells
{{stack,
A ganglion cell is a cell found in a ganglion. Examples of ganglion cells include:
* Retinal ganglion cell (RGC) found in the ganglion cell layer of the retina
* Cells that reside in the adrenal medulla, where they are involved in th ...
, stimulated with light patterns from engineered goggles.
Identification of particular neurons and networks
Amygdala
Optogenetic approaches have been used to map neural circuits in the
amygdala
The amygdala (; plural: amygdalae or amygdalas; also '; Latin from Greek, , ', 'almond', 'tonsil') is one of two almond-shaped clusters of nuclei located deep and medially within the temporal lobes of the brain's cerebrum in complex verteb ...
that contribute to
fear conditioning
Pavlovian fear conditioning is a behavioral paradigm in which organisms learn to predict aversive events. It is a form of learning in which an aversive stimulus (e.g. an electrical shock) is associated with a particular neutral context (e.g., a r ...
.
One such example of a neural circuit is the connection made from the
basolateral amygdala to the dorsal-medial prefrontal cortex where
neuronal oscillations
Neural oscillations, or brainwaves, are rhythmic or repetitive patterns of neural activity in the central nervous system. Neural tissue can generate oscillatory activity in many ways, driven either by mechanisms within individual neurons or by ...
of 4 Hz have been observed in correlation to fear induced freezing behaviors in mice. Transgenic mice were introduced with channelrhodoposin-2 attached with a
parvalbumin
Parvalbumin (PV) is a calcium-binding protein with low molecular weight (typically 9-11 kDa). In humans, it is encoded by the ''PVALB'' gene. It is not a member of the albumin family; it is named for its size (''parv-'', from Latin ''parvus'' smal ...
-Cre promoter that selectively infected interneurons located both in the basolateral amygdala and the dorsal-medial prefrontal cortex responsible for the 4 Hz oscillations. The interneurons were optically stimulated generating a freezing behavior and as a result provided evidence that these 4 Hz oscillations may be responsible for the basic fear response produced by the neuronal populations along the dorsal-medial prefrontal cortex and basolateral amygdala.
Olfactory bulb
Optogenetic activation of olfactory sensory neurons was critical for demonstrating timing in odor processing and for mechanism of neuromodulatory mediated
olfactory
The sense of smell, or olfaction, is the special sense through which smells (or odors) are perceived. The sense of smell has many functions, including detecting desirable foods, hazards, and pheromones, and plays a role in taste.
In humans, it ...
guided behaviors (e.g.
aggression
Aggression is overt or covert, often harmful, social interaction with the intention of inflicting damage or other harm upon another individual; although it can be channeled into creative and practical outlets for some. It may occur either reacti ...
,
mating
In biology, mating is the pairing of either opposite-sex or hermaphroditic organisms for the purposes of sexual reproduction. ''Fertilization'' is the fusion of two gametes. ''Copulation'' is the union of the sex organs of two sexually reproduc ...
) In addition, with the aid of optogenetics, evidence has been reproduced to show that the "afterimage" of odors is concentrated more centrally around the olfactory bulb rather than on the periphery where the olfactory receptor neurons would be located. Transgenic mice infected with channel-rhodopsin Thy1-ChR2, were stimulated with a 473 nm laser transcranially positioned over the dorsal section of the olfactory bulb. Longer photostimulation of
mitral
The mitral valve (), also known as the bicuspid valve or left atrioventricular valve, is one of the four heart valves. It has two cusps or flaps and lies between the left atrium and the left ventricle of the heart. The heart valves are all one-w ...
cells in the olfactory bulb led to observations of longer lasting neuronal activity in the region after the photostimulation had ceased, meaning the olfactory sensory system is able to undergo long term changes and recognize differences between old and new odors.
Nucleus accumbens
Optogenetics, freely moving mammalian behavior, ''in vivo'' electrophysiology, and
slice physiology have been integrated to probe the
cholinergic
Cholinergic agents are compounds which mimic the action of acetylcholine and/or butyrylcholine. In general, the word "choline" describes the various quaternary ammonium salts containing the ''N'',''N'',''N''-trimethylethanolammonium cation. F ...
interneuron
Interneurons (also called internuncial neurons, relay neurons, association neurons, connector neurons, intermediate neurons or local circuit neurons) are neurons that connect two brain regions, i.e. not direct motor neurons or sensory neurons. I ...
s of the
nucleus accumbens
The nucleus accumbens (NAc or NAcc; also known as the accumbens nucleus, or formerly as the ''nucleus accumbens septi'', Latin for "nucleus adjacent to the septum") is a region in the basal forebrain rostral to the preoptic area of the hypotha ...
by direct excitation or inhibition. Despite representing less than 1% of the total population of accumbal neurons, these cholinergic cells are able to control the activity of the
dopamine
Dopamine (DA, a contraction of 3,4-dihydroxyphenethylamine) is a neuromodulatory molecule that plays several important roles in cells. It is an organic compound, organic chemical of the catecholamine and phenethylamine families. Dopamine const ...
rgic terminals that innervate medium spiny neurons (MSNs) in the nucleus accumbens.
These accumbal MSNs are known to be involved in the
neural pathway
In neuroanatomy, a neural pathway is the connection formed by axons that project from neurons to make synapses onto neurons in another location, to enable neurotransmission (the sending of a signal from one region of the nervous system to an ...
through which
cocaine
Cocaine (from , from , ultimately from Quechuan languages, Quechua: ''kúka'') is a central nervous system (CNS) stimulant mainly recreational drug use, used recreationally for its euphoria, euphoric effects. It is primarily obtained from t ...
exerts its effects, because decreasing cocaine-induced changes in the activity of these neurons has been shown to inhibit cocaine
conditioning Conditioning may refer to:
Science, computing, and technology
* Air conditioning, the removal of heat from indoor air for thermal comfort
** Automobile air conditioning, air conditioning in a vehicle
** Ice storage air conditioning, air condition ...
. The few cholinergic neurons present in the nucleus accumbens may prove viable targets for
pharmacotherapy
Pharmacotherapy is therapy using pharmaceutical drugs, as distinguished from therapy using surgery (surgical therapy), radiation (radiation therapy), movement ( physical therapy), or other modes. Among physicians, sometimes the term ''medical the ...
in the treatment of
cocaine dependence
Cocaine dependence is a neurological disorder that is characterized by withdrawal symptoms upon cessation from cocaine use. It also often coincides with cocaine addiction which is a biopsychosocial disorder characterized by persistent use of coc ...
Prefrontal cortex
![Optogenetics imetronic](https://upload.wikimedia.org/wikipedia/commons/7/75/Optogenetics_imetronic.JPG)
''In vivo'' and ''in vitro'' recordings of individual CAMKII AAV-ChR2 expressing
pyramidal neuron
Pyramidal cells, or pyramidal neurons, are a type of multipolar neuron found in areas of the brain including the cerebral cortex, the hippocampus, and the amygdala. Pyramidal neurons are the primary excitation units of the mammalian prefrontal cor ...
s within the prefrontal cortex demonstrated high fidelity action potential output with short pulses of blue light at 20 Hz (Figure 1).
Motor cortex
''In vivo'' repeated optogenetic stimulation in healthy animals was able to eventually induce seizures. This model has been termed optokindling.
Piriform cortex
''In vivo'' repeated optogenetic stimulation of pyramidal cells of the piriform cortex in healthy animals was able to eventually induce seizures.
''In vitro'' studies have revealed a loss of feedback inhibition in the piriform circuit due to impaired GABA synthesis.
Heart
Optogenetics was applied on atrial
cardiomyocytes
Cardiac muscle (also called heart muscle, myocardium, cardiomyocytes and cardiac myocytes) is one of three types of vertebrate muscle tissues, with the other two being skeletal muscle and smooth muscle. It is an involuntary, striated muscle tha ...
to end spiral wave
arrhythmias
Arrhythmias, also known as cardiac arrhythmias, heart arrhythmias, or dysrhythmias, are irregularities in the heartbeat, including when it is too fast or too slow. A resting heart rate that is too fast – above 100 beats per minute in adults ...
, found to occur in
atrial fibrillation, with light.
This method is still in the development stage. A recent study explored the possibilities of optogenetics as a method to correct for arrythmias and resynchronize cardiac pacing. The study introduced channelrhodopsin-2 into cardiomyocytes in ventricular areas of hearts of transgenic mice and performed ''in vitro'' studies of photostimulation on both open-cavity and closed-cavity mice. Photostimulation led to increased activation of cells and thus increased ventricular contractions resulting in increasing heart rates. In addition, this approach has been applied in cardiac resynchronization therapy (
CRT) as a new biological pacemaker as a substitute for electrode based-CRT. Lately, optogenetics has been used in the heart to defibrillate ventricular arrhythmias with local epicardial illumination, a generalized whole heart illumination or with customized stimulation patterns based on arrhythmogenic mechanisms in order to lower defibrillation energy.
Spiral ganglion
Optogenetic stimulation of the
spiral ganglion
The spiral (cochlear) ganglion is a group of neuron cell bodies in the modiolus, the conical central axis of the cochlea. These bipolar neurons innervate the hair cells of the organ of Corti. They project their axons to the ventral and dorsal co ...
in
deaf
Deafness has varying definitions in cultural and medical contexts. In medical contexts, the meaning of deafness is hearing loss that precludes a person from understanding spoken language, an Audiology, audiological condition. In this context it ...
mice restored auditory activity. Optogenetic application onto the
cochlea
The cochlea is the part of the inner ear involved in hearing. It is a spiral-shaped cavity in the bony labyrinth, in humans making 2.75 turns around its axis, the modiolus. A core component of the cochlea is the Organ of Corti, the sensory org ...
r region allows for the stimulation or inhibition of the spiral ganglion cells (SGN). In addition, due to the characteristics of the resting potentials of SGN's, different variants of the protein channelrhodopsin-2 have been employed such as Chronos,
CatCh and f-Chrimson.
Chronos and CatCh variants are particularly useful in that they have less time spent in their deactivated states, which allow for more activity with less bursts of blue light emitted. Additionally, using engineered red-shifted channels as f-Chrimson allow for stimulation using longer wavelengths, which decreases the potential risks of phototoxicity in the long term without compromising gating speed.
The result being that the LED producing the light would require less energy and the idea of cochlear prosthetics in association with photo-stimulation, would be more feasible.
Brainstem
Optogenetic stimulation of a modified red-light excitable channelrhodopsin (ReaChR) expressed in the
facial motor nucleus
The facial motor nucleus is a collection of neurons in the brainstem that belong to the facial nerve ( cranial nerve VII). These lower motor neurons innervate the muscles of facial expression and the stapedius.
Structure
The nucleus is situate ...
enabled minimally invasive activation of
motoneurons
A motor neuron (or motoneuron or efferent neuron) is a neuron whose cell body is located in the motor cortex, brainstem or the spinal cord, and whose axon (fiber) projects to the spinal cord or outside of the spinal cord to directly or indirectly ...
effective in driving whisker movements in mice.
One novel study employed optogenetics on the
Dorsal Raphe Nucleus
The dorsal raphe nucleus is located on the midline of the brainstem and is one of the raphe nuclei. It has rostral and caudal subdivisions.
* The rostral aspect of the ''dorsal'' raphe is further divided into interfascicular, ventral, ventrolatera ...
to both activate and inhibit dopaminergic release onto the ventral tegmental area. To produce activation transgenic mice were infected with channelrhodopsin-2 with a TH-Cre promoter and to produce inhibition the
hyperpolarizing opsin NpHR was added onto the TH-Cre promoter. Results showed that optically activating dopaminergic neurons led to an increase in social interactions, and their inhibition decreased the need to socialize only after a period of isolation.
Visual system
Studying the visual system using optogenetics can be challenging. Indeed, the light used for optogenetic control may lead to the activation of photoreceptors, as a result of the proximity between primary visual circuits and these photoreceptors. In this case, spatial selectivity is difficult to achieve (particularly in the case of the fly optic lobe). Thus, the study of the visual system requires spectral separation, using
channels
Channel, channels, channeling, etc., may refer to:
Geography
* Channel (geography), in physical geography, a landform consisting of the outline (banks) of the path of a narrow body of water.
Australia
* Channel Country, region of outback Austral ...
that are activated by different wavelengths of light than
rhodopsin
Rhodopsin, also known as visual purple, is a protein encoded by the RHO gene and a G-protein-coupled receptor (GPCR). It is the opsin of the rod cells in the retina and a light-sensitive receptor protein that triggers visual phototransduction ...
s within the photoreceptors (peak activation at 480 nm for Rhodopsin 1 in ''
Drosophila
''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
''). Red-shifted CsChrimson or bistable Channelrhodopsin are used for optogenetic activation of neurons (i.e.
depolarization
In biology, depolarization or hypopolarization is a change within a cell, during which the cell undergoes a shift in electric charge distribution, resulting in less negative charge inside the cell compared to the outside. Depolarization is esse ...
), as both allow spectral separation. In order to achieve neuronal silencing (i.e.
hyperpolarization), an anion channelrhodopsin discovered in the cryptophyte algae species ''
Guillardia theta'' (named GtACR1).
can be used. GtACR1 is more light sensitive than other inhibitory channels such as the Halorhodopsin class of chlorid pumps and imparts a strong conductance. As its activation peak (515 nm) is close to that of Rhodopsin 1, it is necessary to carefully calibrate the optogenetic illumination as well as the visual stimulus. The factors to take into account are the wavelength of the optogenetic illumination (possibly higher than the activation peak of GtACR1), the size of the stimulus (in order to avoid the activation of the channels by the stimulus light) and the intensity of the optogenetic illumination. It has been shown that GtACR1 can be a useful inhibitory tool in optogenetic study of ''
Drosophila
''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
''
's visual system by silencing T4/T5 neurons expression. These studies can also be led on intact behaving animals, for instance to probe
optomotor response
In behavioral biology, the optomotor response is an innate, orienting behavior evoked by whole-field visual motion and is common to fish and insects during locomotion, such as swimming, walking and flying. The optomotor response has algorithmic ...
.
Sensorimotor system
Optogenetically inhibiting or activating neurons tests their necessity and sufficiency, respectively, in generating a behavior. Using this approach, researchers can dissect the neural circuitry controlling motor output. By perturbing neurons at various places in the sensorimotor system, researchers have learned about the role of descending neurons in eliciting stereotyped behaviors, how localized tactile sensory input and activity of interneurons alters locomotion, and the role of
Purkinje cells
Purkinje cells, or Purkinje neurons, are a class of GABAergic inhibitory neurons located in the cerebellum. They are named after their discoverer, Czech anatomist Jan Evangelista Purkyně, who characterized the cells in 1839.
Structure
T ...
in generating and modulating movement. This is a powerful technique for understanding the neural underpinnings of
animal locomotion
Animal locomotion, in ethology, is any of a variety of methods that animal (biology), animals use to move from one place to another. Some modes of locomotion are (initially) self-propelled, e.g., running, swimming, jumping, flying, hopping, soari ...
and movement more broadly.
Precise temporal control of interventions
The currently available optogenetic actuators allow for the accurate temporal control of the required intervention (i.e. inhibition or excitation of the target neurons) with precision routinely going down to the millisecond level. The temporal precision varies, however, across optogenetic actuators, and depends on the frequency and intensity of the stimulation.
Experiments can now be devised where the light used for the intervention is triggered by a particular element of behavior (to inhibit the behavior), a particular unconditioned stimulus (to associate something to that stimulus) or a particular oscillatory event in the brain (to inhibit the event). This kind of approach has already been used in several brain regions:
Hippocampus
Sharp waves and ripple complexes (SWRs) are distinct high frequency oscillatory events in the
hippocampus
The hippocampus (via Latin from Greek , 'seahorse') is a major component of the brain of humans and other vertebrates. Humans and other mammals have two hippocampi, one in each side of the brain. The hippocampus is part of the limbic system, a ...
thought to play a role in memory formation and consolidation. These events can be readily detected by following the oscillatory cycles of the on-line recorded
local field potential
Local field potentials (LFP) are transient electrical signals generated in nervous and other tissues by the summed and synchronous electrical activity of the individual cells (e.g. neurons) in that tissue. LFP are "extracellular" signals, meaning ...
. In this way the onset of the event can be used as a trigger signal for a light flash that is guided back into the hippocampus to inhibit neurons specifically during the SWRs and also to optogenetically inhibit the oscillation itself.
These kinds of "closed-loop" experiments are useful to study SWR complexes and their role in memory.
Cellular biology/cell signaling pathways
Analogously to how natural light-gated ion channels such as channelrhodopsin-2 allows optical control of ion flux, which is especially useful in neuroscience, natural light-controlled signal transduction proteins also allow optical control of biochemical pathways, including both second-messenger generation and protein-protein interactions, which is especially useful in studying cell and developmental biology.
In 2002, the first example of using photoproteins from another organism for controlling a biochemical pathway was demonstrated using the light-induced interaction between plant phytochrome and phytochrome-interacting factor (PIF) to control gene transcription in yeast.
By fusing phytochrome to a DNA-binding domain and PIF to a transcriptional activation domain, transcriptional activation of genes recognized by the DNA-binding domain could be induced by light.
This study anticipated aspects of the later development of optogenetics in the brain, for example, by suggesting that "Directed light delivery by fiber optics has the potential to target selected cells or tissues, even within larger, more-opaque organisms."
The literature has been inconsistent as to whether control of cellular biochemistry with photoproteins should be subsumed within the definition of optogenetics, as optogenetics in common usage refers specifically to the control of neuronal firing with opsins,
and as control of neuronal firing with opsins postdates and utilizes distinct mechanisms from control of cellular biochemistry with photoproteins.
Photosensitive proteins utilized in various cell signaling pathways
In addition to phytochromes, which are found in plants and cyanobacteria, LOV domains(
Light-oxygen-voltage-sensing domain A Light-oxygen-voltage-sensing domain (LOV domain) is a protein sensor used by a large variety of higher plants, microalgae, fungi and bacteria to sense environmental conditions. In higher plants, they are used to control phototropism, chloroplast r ...
) from plants and yeast and cryptochrome domains from plants are other natural photosensory domains that have been used for optical control of biochemical pathways in cells.
In addition, a synthetic photosensory domain has been engineered from the fluorescent protein Dronpa for optical control of biochemical pathways.
In photosensory domains, light absorption is either coupled to a change in protein-protein interactions (in the case of phytochromes, some LOV domains, cryptochromes, and Dronpa mutants) or a conformational change that exposes a linked protein segment or alters the activity of a linked protein domain (in the case of phytochromes and some LOV domains).
Light-regulated protein-protein interactions can then be used to recruit proteins to DNA, for example to induce gene transcription or DNA modifications, or to the plasma membrane, for example to activate resident signaling proteins.
CRY2 also clusters when active, so has been fused with signaling domains and subsequently photoactivated to allow for clustering-based activation. The LOV2 domain of ''Avena sativa''(common oat) has been used to expose short peptides or an active protein domain in a light-dependent manner. Introduction of this LOV domain into another protein can regulate function through light induced peptide disorder. The asLOV2 protein, which optogenetically exposes a peptide, has also been used as a scaffold for several synthetic light induced dimerization and light induced dissociation systems (iLID and LOVTRAP, respectively). The systems can be used to control proteins through a protein splitting strategy. Photodissociable Dronpa domains have also been used to cage a protein active site in the dark, uncage it after cyan light illumination, and recage it after violet light illumination.
Temporal control of signal transduction with light
The ability to optically control signals for various time durations is being explored to elucidate how cell signaling pathways convert signal duration and response to different outputs.
Natural signaling cascades are capable of responding with different outputs to differences in stimulus timing duration and dynamics. For example, treating PC12 cells with epidermal growth factor (EGF, inducing a transient profile of ERK activity) leads to cellular proliferation whereas introduction of nerve growth factor (NGF, inducing a sustained profile of ERK activity) leads to differentiation into neuron-like cells. This behavior was initially characterized using EGF and NGF application, but the finding has been partially replicated with optical inputs. In addition, a rapid negative feedback loop in the RAF-MEK-ERK pathway was discovered using pulsatile activation of a photoswitchable RAF engineered with photodissociable Dronpa domains.
Optogenetic noise-photostimulation
Professor Elias Manjarrez´s research group introduced the Optogenetic noise-photostimulation.
This is a technique that uses random noisy light to activate neurons expressing ChR2. An optimal level of optogenetic-noise photostimulation on the brain can increase the somatosensory evoked field potentials, the firing frequency response of pyramidal neurons to somatosensory stimulation, and the sodium current amplitude.
References
Further reading
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External links
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