optogenetics optogenetics optogenetics is a biological technique to control the activity of neurons or other cell types with light. this is achieved by expression of light-sensitive ion channels, pumps or enzymes specifically in the target cells. on the level of individual cells, light-activated enzymes and transcription factors 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 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. 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 of 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 (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, 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 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, 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 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 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, his laboratory staff, including graduate students ed boyden and feng zhang, 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 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. this approach was eventually realized in a human patient by botond roska and coworkers in 2021. 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 (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 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 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, 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. also that year, year bamberg, boyden, deisseroth, hegemann, miesenböck and nagel were jointly awarded the brain prize 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 for "spearheading optogenetics” and "revolutionizing systems neuroscience research." in 2019, bamberg, boyden, deisseroth, hegemann, miesenböck and nagel were awarded the rumford prize by the american academy of arts and sciences 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, 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. description 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. 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 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. 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 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 (chr2, chr1, vchr1, and sfos) 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 (nphr), enhanced halorhodopsins (enphr2.0 and enphr3.0, see figure 2), archaerhodopsin (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 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 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