Optogenetics
Optogenetics most commonly refers to a biological technique that involves the use of light to control neurons that have been genetically modified to express light-sensitive ion channels. As such, optogenetics is a neuromodulation method that uses a combination of techniques from optics and genetics to control the activities of individual neurons in living tissue—even within freely-moving animals. In some usages, optogenetics also refers to optical monitoring of neuronal activity and control of biochemical pathways in non-neuronal cells, although these research activities preceded the use of light-sensitive ion channels in neurons. As optogenetics is used by some authors to refer to only optical control of the activity of genetically defined neurons and not these additional research approaches, the term optogenetics is an example of polysemy.
Neuronal control is achieved using optogenetic actuators like channelrhodopsin, halorhodopsin, and archaerhodopsin, while optical recording of neuronal activities can be made with the help of optogenetic sensors for calcium, vesicular release, neurotransmitters, or membrane voltage. Control of activity is restricted to genetically defined neurons and performed in a spatiotemporal-specific manner by light.
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. At the same time, optogenetics was highlighted in the article on "Breakthroughs of the Decade" in the academic research journal Science.
History
In 1979, Francis Crick suggested that controlling all cells from one type in the brain while leaving the others more or less unaltered is a real challenge for neuroscience. Francis Crick 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 et al. and Ernst Bamberg tried the heterologous expression of microbial rhodopsins and showed light-induced current.
An earlier use of light to activate neurons was carried out by Richard Fork, who demonstrated laser activation of neurons within intact tissue, although not in a genetically-targeted manner. The earliest genetically targeted method that used light to control rhodopsin-sensitized neurons was reported in January 2002, by Boris Zemelman and Gero Miesenböck, who employed Drosophila rhodopsin cultured mammalian neurons. In 2003, Zemelman and Miesenböck developed a second method for light-dependent activation of neurons in which single inotropic 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, 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 produced large photocurents when expressed in the oocyte of a frog. To identify expressing cells, they replaced the cytoplasmic tail of the algal protein with the 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 Ca2+ concentration or to depolarize the cell membrane, simply by illumination".
Karl Deisseroth 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, Karl Deisseroth's laboratory including graduate students Ed Boyden and Feng Zhang published the first demonstration of a single-component optogenetic system, in neurons using the channelrhodopsin-2-eYFP construct from Nagel and Hegemann.
Zhuo-Hua Pan of Wayne State University, 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
In April 2005, Susana Lima and Miesenböck reported the first use of genetically-targeted P2X2 photostimulation to control the behaviour of an animal. They showed that photostimulation of genetically circumscribed groups of neurons, such as those of the dopaminergic 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 and were first to use channelrhodopsin-2 for controlling neuronal activity in an intact animal, showing that motor patterns in the roundworm Caenorhabditis elegans could be evoked by light stimulation of genetically selected neural circuits. In mice, controlled expression of optogenetic tools is often achieved with cell-type-specific Cre/loxP methods developed for neuroscience by Joe Z. Tsien back in the 1990s to activate or inhibit specific brain regions and cell-types in vivo.
In 2007, the labs of Edward Boyden and Karl Deisseroth simultaneously reported successful optogenetic inhibition of activity in neurons.
In 2007, Georg Nagel group and Peter Hegemann group 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. characerized 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.
In parallel to the development of optogentic actuators, optogenetic sensors of activity were developed, for example genetically encoded calcium indicators. The first GECI to be used to image activity in an animal was cameleon, designed by Atsushi Miyawaki, Roger Tsien and coworkers. Cameleon was first used successfully in an animal by Rex Kerr, William Schafer and coworkers to record from neurons and muscle cells of the nematode C. elegans. Cameleon was subsequently used to record neural activity in flies and zebrafish. In mammals, the first GECI to be used in vivo was GCaMP, first developed by Nakai and coworkers. GCaMP has undergone numerous improvements, and GCaMP6 in particular has become widely used throughout neuroscience.
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.
Georg Nagel, Peter Hegemann and Ernst Bamberg were also awarded the Karl Heinz Beckurts Prize in 2010.
In 2010, 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 Georg Nagel, Peter Hegemann, Ernst Bamberg and Deisseroth were awarded the Zülch Prize. In 2012, Miesenböck was awarded the Baillet Latour Health Prize for "pioneering optogenetic approaches to manipulate neuronal activity and to control animal behaviour."
In 2013 Nagel and Peter Hegemann were awarded the Louis-Jeantet Prize for Medicine,.
In 2013, Bamberg, Boyden, Deisseroth, Hegemann, Miesenböck and Nagel were awarded The Brain Prize for "their invention and refinement of optogenetics."
In 2017, Deisseroth was awarded the Else Kröner Fresenius Research Prize 2017 for "his discoveries in optogenetics and hydrogel-tissue chemistry". Deisseroth was named the laureate of the 2018 Kyoto Prize "for the development of optogenetics and causal systems neuroscience" and the 2020 Heineken Prize in Medicine from the Royal Netherlands Academy of Arts and Sciences, for developing optogenetics.
In 2019, Ernst Bamberg,Georg Nagel, Ed Boyden, Karl Deisseroth, Peter Hegemann and Gero Miesenböck were awarded the Rumford Prize for "extraordinary contributions related to the invention and refinement of optogenetics," with.
In 2020, Miesenböck, Hegemann and Georg Nagel jointly received the for "the development of optogenetics".
Description
Optogenetics provides millisecond-scale temporal precision which allows the experimenter to keep pace with fast biological information processing. 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 . By comparison, the temporal precision of traditional genetic manipulations 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 or with reporter proteins that are biosensors, where scientists have fused fluorescent proteins to detector proteins. An example of this is voltage-sensitive fluorescent protein. Additionally, beyond its scientific impact optogenetics represents an important case study in the value of both ecological conservation, and in the importance of pure basic science as these opsins 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 channelrhodopsins to excite neurons and anion-conducting channelrhodopsins for light-induced inhibition. Indirectly light-controlled potassium channels 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, enhanced halorhodopsins, archaerhodopsin, fungal opsins and enhanced bacteriorhodopsin.
Optogenetic control of well-defined biochemical events within behaving mammals is now also possible. Building on prior work fusing vertebrate opsins to specific G-protein coupled receptors 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 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 cyclases 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 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. To stimulate superficial brain areas such as the cerebral cortex, optical fibers or LEDs 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. Recent progress investigate the use of organic LEDs as stimuli for optogenetics. The precise and controlled stimulation of neurons expressing microbial opsin has been demonstrated in vitro on a time scale in the order of a millisecond. Pulsed mode operation allows neural stimulation within compatible low temperature.
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. In invertebrates such as worms and fruit flies some amount of all-trans-retinal 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
The technique of using optogenetics is flexible and adaptable to the experimenter's needs. For starters, experimenters genetically engineer a microbial opsin based on the gating properties required for the experiment.There is a challenge in introducing the microbial opsin, an optogenetic actuator, into a specific region of the organism in question. A rudimentary approach is to introduce an engineered viral vector that contains the optogenetic actuator gene attached to a recognizable promoter such as CAMKIIα. This allows for some level of specificity as cells that already contain and can translate the given promoter will be infected with the viral vector and hopefully express the optogenetic actuator gene.
Another approach is the creation of transgenic mice where the optogenetic actuator gene is introduced into mice zygotes with a given promoter, most commonly Thy1. Introduction of the optogenetic actuator at an early stage allows for a larger genetic code to be incorporated and as a result, increases the specificity of cells to be infected.
A third and rather novel approach that has been developed is creating transgenic mice with Cre recombinase, an enzyme that catalyzes recombination between two lox-P sites. Then by introducing an engineered viral vector containing the optogenetic actuator gene in between two lox-P sites, only the cells containing the Cre recombinase will express the microbial opsin. This last 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, depending on the type of analysis being performed, application of light can be placed at the terminal ends or the main region where the infected cells are situated. Light stimulation can be performed with a vast array of instruments from light emitting diodes or diode-pumped solid state. These light sources are most commonly connected to a computer through a fiber optic cable. Recent advances include the advent of wireless head-mounted devices that also apply LED to targeted areas and as a result give the animal more freedom of mobility to reproduce in vivo results.
Issues
Although already a powerful scientific tool, optogenetics, according to Doug Tischer & Orion D. Weiner of the University of California San Francisco, should be regarded as a "first-generation GFP" because of its immense potential for both utilization and optimization. With that being said, the current approach to optogenetics is limited primarily by its versatility. Even within the field of Neuroscience where it is most potent, the technique is less robust on a subcellular level.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.Moreover, 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 from Type 1 — where neurons operate as integrators — to Type 2 where neurons operate as resonators.
Type 1 excitable media sustain propagating waves of activity whereas Type 2 excitable media do not. The transformation from one to the other explains how constant optical stimulation of primate motor cortex elicits gamma-band oscillations in the manner of a Type 2 excitable medium. Yet those same oscillations propagate far into the surrounding tissue in the manner of a Type 1 excitable medium.
Nonetheless, 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 dendrites, somata or axon terminals would provide a more robust understanding of neuronal circuitry.
Kinetics and synchronization
An issue with channelrhodopsin-2 is that its gating properties don't mimic in vivo cation channels of cortical neurons. A solution to this issue with a protein's kinetic property is introduction of variants of channelrhodopsin-2 with more favorable kinetics.]Another one of the technique's limitations is that light stimulation produces a synchronous activation of infected cells and this removes any individual cell properties of activation among the population affected. Therefore, it is difficult to understand how the cells in the population affected communicate with one another or how their phasic properties of activation may relate to the circuitry being observed.
Optogenetic activation has been combined with functional magnetic resonance imaging to elucidate the connectome, a thorough map of the brain's neural connections. The results, however, are limited by the general properties of fMRI. The readouts from this neuroimaging procedure lack the spatial and temporal resolution appropriate for studying the densely packed and rapid-firing neuronal circuits.
Excitation spectrum
The opsin proteins currently in use have absorption peaks across the visual spectrum, but remain considerable sensitivity to blue light. This spectral overlap makes it very difficult to combine opsin activation with genetically encoded indictors, 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.Additional data indicates that the absorption spectra of organic dyes and fluorescent proteins, used in optogenetics applications, extends from around 250 nm to around 600 nm. Particular organic compounds used in discrete portions of this range include: retinals, flavins, folates, p-coumaric acids, phytochrome chromophotes, cobalamins, and at least six fluorescent proteins including mOrange and mCherry.
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. Moreover, on the clinical side, optogenetics-driven research has led to insights into Parkinson's disease and other neurological and psychiatric disorders. Indeed, optogenetics papers in 2009 have also provided insight into neural codes relevant to autism, Schizophrenia, drug abuse, anxiety, and depression.Identification of particular neurons and networks
Amygdala
Optogenetic approaches have been used to map neural circuits in the amygdala that contribute to fear conditioning. One such example of a neural circuit is the connection made from the basolateral amygdala to the dorsal-medial prefrontal cortex where neuronal oscillations 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-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 guided behaviors 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 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 interneurons of the nucleus accumbens 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 dopaminergic terminals that innervate medium spiny neurons in the nucleus accumbens. These accumbal MSNs are known to be involved in the neural pathway through which cocaine exerts its effects, because decreasing cocaine-induced changes in the activity of these neurons has been shown to inhibit cocaine conditioning. The few cholinergic neurons present in the nucleus accumbens may prove viable targets for pharmacotherapy in the treatment of cocaine dependencePrefrontal cortex
In vivo and in vitro recordings of individual CAMKII AAV-ChR2 expressing pyramidal neurons within the prefrontal cortex demonstrated high fidelity action potential output with short pulses of blue light at 20 Hz.Motor cortex
In vivo repeated optogenetic stimulation in healthy animals was able to eventually induce seizures. This model has been termed optokindling.