Optogenetics (from Greekoptos, meaning "visible") uses light to control neurons which have been genetically sensitised to light. It is a neuromodulation technique employed in neuroscience that uses a combination of techniques from optics and genetics to control and monitor the activities of individual neurons in living tissue—even within freely-moving animals—and to precisely measure the effects of those manipulations in real-time. The key reagents used in optogenetics are light-sensitive proteins. Spatially-precise neuronal control is achieved using optogenetic actuators like channelrhodopsin, halorhodopsin, and archaerhodopsin, while temporally-precise recordings can be made with the help of optogenetic sensors for calcium (Aequorin, Cameleon, GCaMP), chloride (Clomeleon) or membrane voltage (Mermaid).
The earliest approaches were developed and applied by Boris Zemelman and Gero Miesenböck, at the Sloan-Kettering Cancer Center in New York City, and Dirk Trauner, Richard Kramer and Ehud Isacoff at the University of California, Berkeley; these methods conferred light sensitivity but were never reported to be useful by other laboratories due to the multiple components these approaches required. A distinct single-component approach involving microbial opsin genes introduced in 2005 turned out to be widely applied, as described below. Optogenetics is known for the high spatial and temporal resolution that it provides in altering the activity of specific types of neurons to control a subject's behaviour.
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. These journals also referenced recent public-access general-interest video Method of the year video and textual SciAm summaries of optogenetics.
The "far-fetched" possibility of using light for selectively controlling precise neural activity (action potential) patterns within subtypes of cells in the brain was articulated by Francis Crick in his Kuffler Lectures at the University of California in San Diego in 1999. An early use of light to activate neurons was carried out by Richard Fork and later Rafael Yuste, who demonstrated laser activation of neurons within intact tissue, although not in a genetically-targeted manner. The earliest genetically targeted method, which used light to control genetically-sensitised neurons, was reported in January 2002 by Boris Zemelman (now at UT Austin) and Gero Miesenböck, who employed Drosophilarhodopsin photoreceptors for controlling neural activity in 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 caged 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. However, these earlier approaches were not applied outside the original laboratories, likely because of technical challenges in delivering the multiple component parts required.
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 August 2005, Karl Deisseroth's laboratory in the Bioengineering Department at Stanford including graduate students Ed Boyden and Feng Zhang (both now at MIT) published the first demonstration of a single-component optogenetic system, beginning in cultured mammalian neurons. using channelrhodopsin, a single-component light-activated cation channel from unicellular algae), whose molecular identity and principal properties rendering it useful for optogenetic studies had been first reported in November 2003 by Georg Nagel. The groups of Gottschalk and Nagel were the first to extend the usability of Channelrhodopsin-2 for controlling neuronal activity to the intact animal by showing that motor patterns in the roundworm Caenorhabditis elegans could be evoked by targeted expression and stimulation of Channelrhodopsin-2 in selected neural circuits (published in December 2005). Now optogenetics has been routinely combined with brain region-and cell type-specific Cre/loxP genetic methods developed for Neuroscience by Joe Z. Tsien back in 1990s  to activate or inhibit specific brain regions and cell-types in vivo.
In 2010 Karl Deisseroth at Stanford University 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 Gero Miesenböck was awarded the InBev-Baillet Latour International Health Prize for "pioneering optogenetic approaches to manipulate neuronal activity and to control animal behaviour." In 2013 Ernst Bamberg, Ed Boyden, Karl Deisseroth, Peter Hegemann, Gero Miesenböck and Georg Nagel were awarded The Brain Prize for "their invention and refinement of optogenetics."
Fig 1. Channelrhodopsin-2 (ChR2) induces temporally precise blue light-driven activity in rat prelimbic prefrontal cortical neurons. a) In vitro schematic (left) showing blue light delivery and whole-cell patch-clamp recording of light-evoked activity from a fluorescent CaMKllα::ChR2-EYFP expressing pyramidal neuron (right) in an acute brain slice. b) In vivo schematic (left) showing blue light (473 nm) delivery and single-unit recording. (bottom left) Coronal brain slice showing expression of CaMKllα::ChR2-EYFP in the prelimbic region. Light blue arrow shows tip of the optical fiber; black arrow shows tip of the recording electrode (left). White bar, 100 µm. (bottom right) In vivo light recording of prefrontal cortical neuron in a transduced CaMKllα::ChR2-EYFP rat showing light-evoked spiking to 20 Hz delivery of blue light pulses (right). Inset, representative light-evoked single-unit response.
Fig 2. Halorhodopsin (NpHR) rapidly and reversibly silences spontaneous activity in vivo in rat prelimbic prefrontal cortex. (Top left) Schematic showing in vivo green (532 nm) light delivery and single- unit recording of a spontaneously active CaMKllα::eNpHR3.0- EYFP expressing pyramidal neuron. (Right) Example trace showing that continuous 532 nm illumination inhibits single-unit activity in vivo. Inset, representative single unit event; Green bar, 10 seconds.
A nematode expressing the light-sensitive ion channel Mac. Mac is a proton pump originally isolated in the fungus Leptosphaeria maculans and now expressed in the muscle cells of C. elegans that opens in response to green light and causes hyperpolarizing inhibition. Of note is the extension in body length that the worm undergoes each time it is exposed to green light, which is presumably caused by Mac's muscle-relaxant effects.
A nematode expressing ChR2 in its gubernacular-oblique muscle group responding to stimulation by blue light. Blue light stimulation causes the gubernacular-oblique muscles to repeatedly contract, causing repetitive thrusts of the spicule, as would be seen naturally during copulation.
Millisecond-scale temporal precision is central to optogenetics, which allows the experimenter to keep pace with fast biological information processing (for example, in probing the causal role of specific action potential 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 biosensors, where scientists have fused fluorescent proteins to detector proteins. An example of this is voltage-sensitive fluorescent protein (VSFP2).
The hallmark of optogenetics therefore is introduction of fast light-activated channels 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 (ChR2, ChR1, VChR1, and SFOs) to excite neurons. For silencing, halorhodopsin (NpHR), enhanced halorhodopsins (eNpHR2.0 and eNpHR3.0), archaerhodopsin (Arch), Leptosphaeria maculans fungal opsins (Mac), and enhanced bacteriorhodopsin (eBR) have been employed to inhibit neurons (see Figure 2), including in freely-moving mammals.
Moreover, 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 (crucially, with tools that displayed low activity in the dark) followed soon thereafter, when optical control over small GTPases and adenylyl cyclases was achieved in cultured cells using novel strategies from several different laboratories. This emerging repertoire of optogenetic probes now allows cell-type-specific and temporally precise control of multiple axes of cellular function within intact animals.
Optogenetics also necessarily includes 1) 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), and 2) 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.
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 vertebrates. In invertebrates such as worms and fruit flies some amount of retinal isomerase all-trans-retinal (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.
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 (see references from the scientific literature below). 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.
It has been pointed out that beyond its scientific impact, optogenetics also 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 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).
Optogenetic activation and/or silencing has been used at least in the following regions:
The Deisseroth laboratory integrated optogenetics, freely moving mammalian behavior, in vivo electrophysiology, and slice physiology to probe the cholinergicinterneurons 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 (MSNs) 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 dependence
In vivo and in vitro recordings (by the Cooper laboratory) 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 (Figure 1). The same group recorded complete green light-induced silencing of spontaneous activity in the same prefrontal cortical neuronal population expressing an AAV-NpHR vector (Figure 2).
^Mancuso, J. J.; Kim, J.; Lee, S.; Tsuda, S.; Chow, N. B. H.; Augustine, G. J. (2010). "Optogenetic probing of functional brain circuitry". Experimental Physiology96 (1): 26–33. doi:10.1113/expphysiol.2010.055731. PMID21056968. edit
^ abZemelman, B. V.; Nesnas, N.; Lee, G.A.; Miesenböck, G. (2003). "Photochemical gating of heterologous ion channels: Remote control over genetically designated populations of neurons". PNAS100: 1352–7. doi:10.1016/S0896-6273(01)00574-8. PMID11779476.
^ abcWitten, I. B.; Lin, S. C.; Brodsky, M.; Prakash, R.; Diester, I.; Anikeeva, P.; Gradinaru, V.; Ramakrishnan, C.; Deisseroth, K. (2010). "Cholinergic interneurons control local circuit activity and cocaine conditioning" Science 330 (6011) 1677–81. . PMC 3142356.doi:10.1126/science.1193771PMID 21164015
^Kim, J. M.; Hwa, J.; Garriga, P.; Reeves, P. J.; RajBhandary, U. L.; Khorana, H. G. (2005). "Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops". Biochemistry44 (7): 2284–92. doi:10.1021/bi048328i. PMID15709741.
^Yazawa, M.; Sadaghiani, A. M.; Hsueh, B.; Dolmetsch, R. E. (2009). "Induction of protein-protein interactions in live cells using light". Nature Biotechnology27 (10): 941–5. doi:10.1038/nbt.1569. PMID19801976.
^Gradinaru, V.; Thompson, K. R.; Zhang, F.; Mogri, M.; Kay, K.; Schneider, M. B.; Deisseroth, K. (2007). "Targeting and readout strategies for fast optical neural control in vitro and in vivo". J. Neurosci27 (52): 14231–8. doi:10.1523/JNEUROSCI.3578-07.2007. PMID18160630.
^Gradinaru, V.; Mogri, M.; Thompson, K. R.; Henderson, J. M.; Deisseroth, K. (2009). "Optical Deconstruction of Parkinsonian Neural Circuitry". Science324 (5925): 354–359. doi:10.1126/science.1167093. PMID19299587. edit
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